Biomarkers for Inflammatory Bowel Disease and Irritable Bowel Syndrome

ABSTRACT

The present invention provides compositions and their use in diagnosing and/or distinguishing inflammatory bowel disease and irritable bowel syndrome.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 61/036,632 filed Mar. 14, 2008 and 61/097,109 filed Sep. 15,2008, both references incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The invention relates generally to the fields of nucleic acids, nucleicacid detection, and intestinal disorders

BACKGROUND

Irritable Bowel Syndrome (IBS) is a chronic condition characterized byabdominal pain, constipation and/or diarrhea. It is fairly common andresponsible for 20-50% of visits to gastroenterologists. IBS does notresult in damage to the gastrointestinal tract and is not associatedwith nor develops into more serious gastrointestinal conditions such ascolon cancer or colitis, and it can often be controlled with drugs aimedat symptom relief, diet changes, and stress management techniques.However, more serious conditions must be ruled out before making adefinitive diagnosis of IBS, and thus setting an appropriate course oftreatment.

Inflammatory Bowel Disease (IBD) refers to at least two distinctdiseases that cause inflammation of the intestinal tract: UlcerativeColitis affects the colon, while Crohn's Disease most often affects thelast part of the small intestine, but can attack any part of thedigestive tract. IBD is rare by comparison to IBS, and IBD patients mayexperience many of the same symptoms as IBS patients, complicating thediagnosis of IBD. Furthermore, patients with IBD are at greater risk ofdeveloping colon cancer. Currently, IBD can only be definitivelydiagnosed by colonoscopy, a rather invasive procedure; even thisinvasive procedure is incapable of diagnosing approximately 10% ofpatients undergoing colonoscopy (Burczynski, J. Mol. Diag. 8(1): 51(2006)). IBD may be treated with anti-inflammatory drugs,immunosuppressive agents, and/or surgery to remove damaged tissues.

Thus, there is a need in the art for better and more specific diagnostictests capable of distinguishing between IBD and IBS.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides biomarkers consistingof between 2 and 35 different nucleic acid probe sets, wherein:

(a) a first probe set that selectively hybridizes under high stringencyconditions to a nucleic acid selected from the group consisting of SEQID NO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ IDNO:14 (PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQID NO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:16 (HMGB1), and SEQ ID NO:6 (CALM3); and

(b) a second probe set that selectively hybridizes under high stringencyconditions to a nucleic acid selected from the group consisting of SEQID NO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ IDNO:14 (PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQID NO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:16 (HMGB1), and SEQ ID NO:6 (CALM3),

wherein the first probe set and the second probe set do not selectivelyhybridize to the same nucleic acid.

In a second aspect, the present invention provides biomarker,comprising:

(a) a first primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ ID NO:14(PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQ IDNO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQ IDNO:16 (HMGB1), and SEQ ID NO:6 (CALM3); and

(b) a second primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ ID NO:14(PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQ IDNO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQ IDNO:16 (HMGB1), and SEQ ID NO:6 (CALM3),

wherein the first primer pair and the second primer pair do notselectively amplify the same nucleic acid.

In a third aspect, the present invention provides methods for diagnosingIBD and/or IBS comprising:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD or IBS under hybridizing conditions with2 or more probes sets, wherein at least a first probe set and a secondprobe set selectively hybridize under high stringency conditions to anucleic acid target selected from the group consisting of SEQ ID NO:4(TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ ID NO:14(PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQ IDNO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQ IDNO:16 (HMGB1), and SEQ ID NO:6 (CALM3); wherein the first probe set andthe second probe set do not selectively hybridize to the same nucleicacid;

(b) detecting formation of hybridization complexes between the 2 or moreprobe sets and nucleic acid targets in the nucleic acid sample, whereina number of such hybridization complexes provides a measure of geneexpression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD, IBS, orneither based on the gene expression of the nucleic acid targets.

In one embodiment of the third aspect of the invention, diagnosingwhether the subject is likely to have IBD, IBS, or neither comprisesanalyzing gene expression of the nucleic acid targets by applying aweight to the number of hybridization complexes formed for each nucleicacid target.

In a fourth aspect, the present invention provides methods fordiagnosing IBD and/or IBS comprising:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD or IBS under amplifying conditions with2 or more primer pairs, wherein at least a first primer pair and asecond primer pair are capable of selectively amplifying a detectableportion of a nucleic acid target selected from the group consisting ofSEQ ID NO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ IDNO:14 (PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQID NO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:16 (HMGB1), and SEQ ID NO:6 (CALM3); wherein the first primer pairand the second primer pair do not selectively amplify the same nucleicacid;

(b) detecting amplification products generated by amplification ofnucleic acid targets in the nucleic acid sample by the two or moreprimer pairs, wherein the amplification products provide a measure ofgene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD, IBS, orneither based on the amplification of the nucleic acid targets.

In one embodiment of the fourth aspect of the invention, diagnosingwhether the subject is likely to have IBD, IBS, or neither based on theamplification of the nucleic acid targets comprises analyzing theamplification products by applying a weight to the number ofamplification products formed for each nucleic acid target.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the weighted-sum regression score for each trainingset patient in an IBD vs. Normal analysis.

FIG. 2 shows the boxplots of the logistic regression scores based onexpression levels of 5 of the 8 genes for the five patient cohorts. Notethe separation of the normal and IBD patients (UC and Crohn's).

FIG. 3 shows the boxplots of the logistic regression scores based onexpression levels of 7 of the 8 genes for the five patient cohorts. Notethe separation of the normal and IBS from the IBD patients (UC andCrohn's).

FIG. 4 shows the boxplots of the logistic regression scores based onexpression levels of 4 of the 8 genes for the five patient cohorts. Notethe separation of the Normal and IBS patients.

FIG. 5 is a boxplot of the logistic regression scores based onexpression levels of 5 genes whose combination differentiates IBD vs.Normal.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

In a first aspect, the invention provides biomarkers consisting ofbetween 2 and 35 different nucleic acid probe sets, wherein:

(a) a first probe set that selectively hybridizes under high stringencyconditions to a nucleic acid selected from the group consisting of SEQID NO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ IDNO:14 (PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQID NO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:16 (HMGB1), and SEQ ID NO:6 (CALM3); and

(b) a second probe set that selectively hybridizes under high stringencyconditions to a nucleic acid selected from the group consisting of SEQID NO:4 (TH1L), SEQ ID NO:11 (HIST1H2AC), SEQ ID NO:12 (TFE3), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:13 (NONO), SEQ IDNO:14 (PCBP1), SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:10 (PPP2R5A), SEQID NO:15 (PGRMC1), SEQ ID NO:3 (BLCAP), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:16 (HMGB1), and SEQ ID NO:6 (CALM3), wherein the first probe setand the second probe set do not selectively hybridize to the samenucleic acid.

The recited nucleic acids are human nucleic acids recited by SEQ ID NOand gene name; as will be understood by those of skill in the art, suchhuman nucleic acid sequences also include the mRNA counterpart to thesequences disclosed herein. For ease of reference, the nucleic acidswill be referred to by gene name throughout the rest of thespecification; it will be understood that as used herein the gene namemeans the recited SEQ. ID. NO(S). for each gene listed in Table 1,complements thereof, and RNA counterparts thereof.

In one non-limiting example, the first probe set selectively hybridizesunder high stringency conditions to TH1L, and thus selectivelyhybridizes under high stringency conditions to the nucleic acid of SEQID NO:4 (NM_(—)198978) a mRNA version thereof, or complements thereof,and the second probe set selectively hybridizes under high stringencyconditions to RAP1A, thus selectively hybridizing under high stringencyconditions to one or both of the nucleic acid of SEQ ID NOS: 1-2, a mRNAversion thereof, or complements thereof. Further embodiments will bereadily apparent to those of skill in the art based on the teachingsherein and Table 1 below.

TABLE 1 Nucleic acid sequences NCBI SEQ Gene name accession # ID # Hs.IDgene description TH1L NM_198976 4 Hs.517148 TH1-like (Drosophila)HIST1H2AC NM_003512 11 Hs.484950 histone 1, H2ac TFE3 NM_006521 12Hs.274184 Transcription factor binding to IGHM enhancer 3 HIST1H2BKNM_080593 9 Hs.437275 Histone 1, H2bk UBE2G1 NM_003342.4 5 Hs.462035Ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, C. elegans) NONONM_007363 13 Hs.533282 Non-POU domain containing, octamer- binding PCBP1NM_006196 14 Hs.2853 Poly(rC) binding protein 1 RAP1A (1) NM_001010935.112 Hs.190334 RAP1A, member of RAS oncogene family RAP1A (2) NM_002884.2PPP2R5A NM_006243 10 Hs.497684 Protein phosphatase 2, regulatory subunitB (B56), alpha isoform PGRMC1 NM_006667 15 Hs.90061 Progesteronereceptor membrane component 1 BLCAP NM_006698 3 Hs.472651 Bladder cancerassociated protein GPX1 (1) NM_000581.2 7 Hs.76686 Glutathioneperoxidase 1 GPX1 (2) NM_201397.1 8 HMGB1 NM_002128 16 Hs.434102High-mobility group box 1 CALM3 NM_005184 6 Hs.515487 Calmodulin 3(phosphorylase kinase, delta)

As is described in more detail below, the inventors have discovered thatthe biomarkers of the invention can be used, for example, as probes fordiagnosing IBD and IBS. The biomarkers can be used, for example, todetermine the expression levels in tissue mRNA for the recited genes.The biomarkers of this first aspect of the invention are especiallypreferred for use in RNA expression analysis from the genes in a tissueof interest, such as blood samples (for example, peripheral bloodmononuclear cells (PBMCs) or RBC-depleted whole blood).

As used herein with respect to all aspects and embodiments of theinvention, a “probe set” is one or more isolated polynucleotides thateach selectively hybridize under high stringency conditions to the sametarget nucleic acid (for example, a single specific mRNA). Thus, asingle “probe set” may comprise any number of different isolatedpolynucleotides that selectively hybridize under high stringencyconditions to the same target nucleic acid, such as a mRNA expressionproduct. For example, a probe set that selectively hybridizes to a BLCAPmRNA may consist of a single polynucleotide of 100 nucleotides thatselectively hybridizes under high stringency conditions to BLCAP mRNA,may consist of two separate polynucleotides 100 nucleotides in lengththat each selectively hybridize under high stringency conditions toBLCAP mRNA, or may consist of twenty separate polynucleotides 25nucleotides in length that each selectively hybridize under highstringency conditions to BLCAP mRNA (such as, for example, fragmenting alarger probe into many individual shorter polynucleotides). Those ofskill in the art will understand that many such permutations arepossible.

The biomarkers of the invention consist of between 2 and 35 probe sets.In various embodiments, the biomarker can include 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 probe sets that selectively hybridize under highstringency conditions to a nucleic acid selected from the groupconsisting of TH1L, HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1,RAP1A, PPP2R5A, PGRMC1, BLCAP, GPX1, HMGB1, and CALM3, wherein each ofthe 3-14 different probe sets selectively hybridize under highstringency conditions to a different nucleic acid target. Thus, as willbe clear to those of skill in the art, the biomarkers may includefurther probe sets that, for example, (a) are additional probe sets thatalso selectively hybridize under high stringency conditions to therecited human nucleic acid (e.g. RAP1A expresses alternative productsthat share much overlap; thus, one probe set could selectively hybridizeto one transcript (or a cDNA derived therefrom) and another probe setcould selectively hybridize to the other alternative transcript (or acDNA derived therefrom; GPX is a similar example with alternativetranscripts); or (b) do not selectively hybridize under high stringencyconditions to any of the recited human nucleic acids. Such further probesets of type (b) may include those consisting of polynucleotides thatselectively hybridize to other nucleic acids of interest, and mayfurther include, for example, probe sets consisting of controlsequences, such as competitor nucleic acids, sequences to provide astandard of hybridization for comparison, etc.

In various embodiments of this first aspect, the biomarker consists of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 probe sets. Invarious further embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of thedifferent probe sets selectively hybridize under high stringencyconditions to a nucleic acid selected from the group consisting of TH1L,HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1,BLCAP, GPX1, HMGB1, and CALM3. As will be apparent to those of skill inthe art, as the percentage of probe sets that selectively hybridizeunder high stringency conditions to a nucleic acid selected from thegroup consisting of TH1L, HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO,PCBP1, RAP1A, PPP2R5A, PGRMC1, BLCAP, GPX1, HMGB1, and CALM3 increases,the maximum number of probe sets in the biomarker will decreaseaccordingly. Thus, for example, where at least 50% of the probe setsselectively hybridize under high stringency conditions to a nucleic acidselected from the group consisting of TH1L, HIST1H2AC, TFE3, HIST1H2BK,UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1, BLCAP, GPX1, HMGB1, andCALM3, or their complements, the biomarker will consist of between 2 and28 probe sets. Those of skill in the art will recognize the variousother permutations encompassed by the compositions according to thevarious embodiments of this aspect of the invention.

As used herein with respect to each aspect and embodiment of theinvention, the term “selectively hybridizes” means that the isolatedpolynucleotides are fully complementary to at least a portion of theirnucleic acid target so as to form a detectable hybridization complexunder the recited hybridization conditions, where the resultinghybridization complex is distinguishable from any hybridization thatmight occur with other nucleic acids. The specific hybridizationconditions used will depend on the length of the polynucleotide probesemployed, their GC content, as well as various other factors as is wellknown to those of skill in the art. (See, for example, Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (“Tijssen”)). As used herein,“stringent hybridization conditions” are selected to be nor more than 5°C. lower than the thermal melting point (Tm) for the specificpolynucleotide at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. High stringencyconditions are selected to be equal to the Tm for a particularpolynucleotide probe. An example of stringent conditions are those thatpermit selective hybridization of the isolated polynucleotides to thegenomic or other target nucleic acid to form hybridization complexes in0.2×SSC at 65° C. for a desired period of time, and wash conditions of0.2×SSC at 65° C. for 15 minutes. It is understood that these conditionsmay be duplicated using a variety of buffers and temperatures. SSC (see,e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989) is wellknown to those of skill in the art, as are other suitable hybridizationbuffers.

The polynucleotides in the probe sets can be of any length that permitsselective hybridization under high stringency conditions to the nucleicacid of interest. In various preferred embodiments of this aspect of theinvention and related aspects and embodiments disclosed below, theisolated polynucleotides are at least 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more contiguousnucleotides in length of one of the recited SEQ ID NOS., fullcomplements thereof, or corresponding RNA sequences.

The term “polynucleotide” as used herein refers to DNA or RNA,preferably DNA, in either single- or double-stranded form. In apreferred embodiment, the polynucleotides are single stranded nucleicacids that are “anti-sense” to the recited nucleic acid (or itscorresponding RNA sequence). The term “polynucleotide” encompassesnucleic-acid-like structures with synthetic backbones. DNA backboneanalogues provided by the invention include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs), methylphosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36:8692-8698), and benzylphosphonate linkages, as discussedin U.S. Pat. No. 6,664,057; see also Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press).

An “isolated” polynucleotide as used herein for all of the aspects andembodiments of the invention is one which is free of sequences whichnaturally flank the polynucleotide in the genomic DNA of the organismfrom which the nucleic acid is derived, and preferably free from linkersequences found in nucleic acid libraries, such as cDNA libraries.Moreover, an “isolated” polynucleotide is substantially free of othercellular material, gel materials, and culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. The polynucleotides of theinvention may be isolated from a variety of sources, such as by PCRamplification from genomic DNA, mRNA, or cDNA libraries derived frommRNA, using standard techniques; or they may be synthesized in vitro, bymethods well known to those of skill in the art, as discussed in U.S.Pat. No. 6,664,057 and references disclosed therein. Syntheticpolynucleotides can be prepared by a variety of solution or solid phasemethods. Detailed descriptions of the procedures for solid phasesynthesis of polynucleotide by phosphite-triester, phosphotriester, andH-phosphonate chemistries are widely available. (See, for example, U.S.Pat. No. 6,664,057 and references disclosed therein). Methods to purifypolynucleotides include native acrylamide gel electrophoresis, andanion-exchange HPLC, as described in Pearson (1983) J. Chrom.255:137-149. The sequence of the synthetic polynucleotides can beverified using standard methods.

In one embodiment, the polynucleotides are double or single strandednucleic acids that include a strand that is “anti-sense” to all or aportion of the SEQ ID NOS shown above for each gene of interest or itscorresponding RNA sequence (ie: it is fully complementary to the recitedSEQ ID NOs). In one non-limiting example, the first probe setselectively hybridizes under high stringency conditions to TH1L, and isfully complementary to all or a portion of the nucleic acid of SEQ IDNO:4 (NM_(—)198978) or a mRNA version thereof, and the second probe setselectively hybridizes under high stringency conditions to RAP1A and isfully complementary to one or both of the nucleic acid of SEQ ID NOS:1-2 or a mRNA version thereof.

In one preferred embodiment of the first aspect of the invention, thebiomarker comprises or consists of at least 3, 4, or 5 probe sets thatselectively hybridize under high stringency conditions to a nucleic acidselected from the group consisting of TH1L, HIST1H2BK, UBE2G1, BLCAP,and CALM3. As disclosed in more detail below, such probe sets can beused in preferred embodiments of the methods of the invention fordistinguishing normal subjects from those subjects suffering from IBS.Examples of such preferred probe sets are provided in Table 17. Thus, ina preferred embodiment, the invention provides a biomarker consisting ofbetween 3 and 35 different nucleic acid probe sets, wherein:

(a) a first probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:4 (TH1L), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ ID NO:6(CALM3), or a full complement thereof;

(b) a second probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:4 (TH1L), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ ID NO:6(CALM3), or a full complement thereof; and

(c) a third probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:4 (TH1L), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ ID NO:6(CALM3), or a full complement thereof; wherein each of the between 3 and35 different probe sets consists of one or more probes of 15 or morecontiguous nucleotides, or full complements thereof, of a single mRNAdifferent from that of the other probe sets, and wherein each of thedifferent probe sets is optionally detectably labeled. In a furtherpreferred version of this embodiment, the first probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofBLCAP, or a full complement thereof; the second probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofTH1L, or a full complement thereof; the third probe set consists of oneor more nucleotide probes of 15 or more contiguous nucleotides ofUBE2G1, or a full complement thereof; and a fourth probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofHIST1H2BK, or a full complement thereof.

In another preferred embodiment of the first aspect of the invention,the biomarker comprises or consists of at least 5 or 6 probe sets thatprobe sets selectively hybridize under high stringency conditions to anucleic acid selected from the group consisting of RAP1A, BLCAP, UBE2G1,GPX1, CALM3, and NONO. As disclosed in more detail below, such probesets can be used in preferred embodiments of the methods of theinvention for distinguishing normal subjects from those subjectssuffering from IBD. Examples of such preferred probe sets are providedin Table 18. In one preferred version of this embodiment, the biomarkerconsists of between 5 and 35 different nucleic acid probe sets, wherein:

(a) a first probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof;

(b) a second probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof;

(c) a third probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof;

(d) a fourth probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof;and

(e) a fifth probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof;

wherein each of the between 5 and 35 different probe sets consists ofone or more probes of 15 or more contiguous nucleotides, or fullcomplements thereof, of a single mRNA different from that of the otherprobe sets, and wherein each of the different probe sets is optionallydetectably labeled. In another version, the first probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofRAP1A, or a full complement thereof; the second probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofBLCAP, or a full complement thereof; the third probe set consists of oneor more nucleotide probes of 15 or more contiguous nucleotides ofUBE2G1, or a full complement thereof; the fourth probe set consists ofone or more nucleotide probes of 15 or more contiguous nucleotides ofCALM3, or a full complement thereof; the fifth probe set consists of oneor more nucleotide probes of 15 or more contiguous nucleotides of GPX1,or a full complement thereof; and a sixth probe set consists of one ormore nucleotide probes of 15 or more contiguous nucleotides of NONO or afull complement thereof.

In a further preferred embodiment of the first aspect of the invention,the biomarker comprises or consists of at least 6 or 7 probe sets thatselectively hybridize under high stringency conditions to a nucleic acidselected from the group consisting of RAP1A, BLCAP, UBE2G1, CALM3, GPX1,HIST1H2BK, and PPP2R5A. As disclosed in more detail below, such probesets can be used in preferred embodiments of the methods of theinvention for distinguishing those subjects suffering from IBS fromthose subjects suffering from IBD. Examples of such preferred probe setsare provided in Table 19. In one preferred embodiment, the biomarkerconsists of

(a) a first probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof;

(b) a second probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof;

(c) a third probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof;

(d) a fourth probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof;

(e) a fifth probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof; and

(f) a sixth probe set consisting of one or more nucleotide probesconsisting of 15 or more contiguous nucleotides of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), ora full complement thereof;

wherein each of the between 6 and 35 different probe sets consists ofone or more probes of 15 or more contiguous nucleotides, or fullcomplements thereof, of a single mRNA different from that of the otherprobe sets, and wherein each of the different probe sets is optionallydetectably labeled. In a further preferred embodiment, the first probeset consists of one or more nucleotide probes of 15 or more contiguousnucleotides of RAP1A, or a full complement thereof; the second probe setconsists of one or more nucleotide probes of 15 or more contiguousnucleotides of UBE2G1, or a full complement thereof; the third probe setconsists of one or more nucleotide probes of 15 or more contiguousnucleotides of CALM3, or a full complement thereof; the fourth probe setconsists of one or more nucleotide probes of 15 or more contiguousnucleotides of GPX1, or a full complement thereof; the fifth probe setconsists of one or more nucleotide probes of 15 or more contiguousnucleotides of HIST1H2BK, or a full complement thereof; the sixth probeset consists of one or more nucleotide probes of 15 or more contiguousnucleotides of PPP2R5A, or a full complement thereof; and a seventhprobe set consists of one or more nucleotide probes of 15 or morecontiguous nucleotides of BLCAP, or a full complement thereof.

In one specific embodiment of this first aspect of the invention, thebiomarker includes a first probe set that selectively hybridizes underhigh stringency conditions to BLCAP, a second probe set that selectivelyhybridizes under high stringency conditions to TH1L, a third probe setthat selectively hybridizes under high stringency conditions to CALM3,and a fourth probe set that selectively hybridizes under high stringencyconditions to HIST1H2BK. As disclosed in more detail below, theinventors have discovered that such biomarkers can be used as probes todistinguish between normal and IBS patients.

In a second specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to BLCAP, a second probe set thatselectively hybridizes under high stringency conditions to TH1L, a thirdprobe set that selectively hybridizes under high stringency conditionsto UBE2G1, and a fourth probe set that selectively hybridizes under highstringency conditions to HIST1H2BK. As disclosed in more detail below,the inventors have discovered that such biomarkers can be used as probesto distinguish between normal and IBS patients.

In a third specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1. As disclosed in more detailbelow, the inventors have discovered that such biomarkers can be used asprobes to distinguish between normal and IBD patients.

In a fourth specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, and a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1. Asdisclosed in more detail below, the inventors have discovered that suchbiomarkers can be used as probes to distinguish between normal and IBDpatients.

In a fifth specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1, and asixth probe set that selectively hybridizes under high stringencyconditions to NONO. As disclosed in more detail below, the inventorshave discovered that such biomarkers can be used as probes todistinguish between normal and IBD patients.

In a sixth specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1, a sixthprobe set that selectively hybridizes under high stringency conditionsto HIST1H2BK, and a seventh probe set that selectively hybridizes underhigh stringency conditions to PPP2RR5A. As disclosed in more detailbelow, the inventors have discovered that such biomarkers can be used asprobes to distinguish normal and IBS patients from IBD patients.

In a seventh specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to UBE2G1, athird probe set that selectively hybridizes under high stringencyconditions to CALM3, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1, a fifth probe set thatselectively hybridizes under high stringency conditions to HIST1H2BK1,and a sixth probe set that selectively hybridizes under high stringencyconditions to PPP2R5A. As disclosed in more detail below, the inventorshave discovered that such biomarkers can be used as probes todistinguish IBS patients from IBD patients.

In an eighth specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to RAP1A, a second probe set thatselectively hybridizes under high stringency conditions to UBE2G1, athird probe set that selectively hybridizes under high stringencyconditions to CALM3, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1, a fifth probe set thatselectively hybridizes under high stringency conditions to HIST1H2BK, asixth probe set that selectively hybridizes under high stringencyconditions to PPP2R5A, and a seventh probe set that selectivelyhybridizes under high stringency conditions to BLCAP. As disclosed inmore detail below, the inventors have discovered that such biomarkerscan be used as probes to distinguish IBS patients from IBD patients.

In a ninth specific embodiment of this first aspect of the invention,the biomarker includes a first probe set that selectively hybridizesunder high stringency conditions to BLCAP, a second probe set thatselectively hybridizes under high stringency conditions to CALM3, athird probe set that selectively hybridizes under high stringencyconditions to GPX1, a fourth probe set that selectively hybridizes underhigh stringency conditions to TH1L, a fifth probe set that selectivelyhybridizes under high stringency conditions to RAP1A, and a sixth probeset that selectively hybridizes under high stringency conditions toNONO. As disclosed in more detail below, the inventors have discoveredthat such biomarkers can be used as probes to distinguish IBS and IBDpatients from normal patients.

Thus, in various other embodiments of this first aspect of theinvention, the biomarker comprises or consists of one or more of:

(a) a first probe set that selectively hybridizes under high stringencyconditions to RAP1A and a second probe set that selectively hybridizesunder high stringency conditions to GPX1 (used as a probe set paircommon to biomarkers for diagnosing IBD from normal patients; IBD fromnormal and IBS patients; IBD from IBS patients; and IBD and IBS patientsfrom normal patients);

(b) a first probe set that selectively hybridizes under high stringencyconditions to UBE2G1 and a second probe set that selectively hybridizesunder high stringency conditions to BLCAP (used as a probe set pair thatcan be added to probe set pair (a) to generate a 4-gene biomarkerspecific for diagnosing IBD from normal);

(c) a first probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a second probe set that selectively hybridizesunder high stringency conditions to BLCAP and a third probe set thatselectively hybridizes under high stringency conditions to CALM3 (usedas a probe trio that can be added to probe set pair (a) to generate a5-gene biomarker for diagnosing IBD from normal);

(d) a first probe set that selectively hybridizes under high stringencyconditions to NONO and a second probe set that selectively hybridizesunder high stringency conditions to CALM3 (used as a probe set pairthat, can be added to probe sets (a) and (b) to generate a 6-genebiomarker for diagnosing IBD from normal);

(e) a first probe set that selectively hybridizes under high stringencyconditions to PPP2R5A and a second probe set that selectively hybridizesunder high stringency conditions to HIST1H2BK (used as a probe set paircommon to biomarkers for diagnosing IBD from IBS patients). The probeset (e), when combined with probe sets (a), and (c) disclosed above fordistinguishing IBD from normal, generates a biomarker for diagnosingnormal and IBS patients from IBD patients). The probe set combination of(e), (a), and (c) can also be used as a biomarker to distinguish betweenIBS and IBD patients when the expression values of the individual genesare given different weights as discussed herein;

(f) a first probe set that selectively hybridizes under high stringencyconditions to UBE2G1 and a second probe set that selectively hybridizesunder high stringency conditions to CALM3 (used as a probe set pair thatcan be added to probe sets (a), and (e) described above, to generate a6-gene biomarker that can be used to distinguish IBD from IBS patients);

(g) a first probe set that selectively hybridizes under high stringencyconditions to BLCAP and a second probe set that selectively hybridizesunder high stringency conditions to TH1L (used as probe set pair thatwhen added to (a) and (d) above can be used to generate a 6-genebiomarker that can distinguish patients with IBS or IBD from normalpatients);

h) a first probe set that selectively hybridizes under high stringencyconditions to HIST1H2 KB and a second probe set that selectivelyhybridizes under high stringency conditions to CALM3; (used as probe setpair that when added to (g) above can be used to generate a 4-genebiomarker that can distinguish patients with IBS from normal patients);and

i) a first probe set that selectively hybridizes under high stringencyconditions to HIST1H2 KB and a second probe set that selectivelyhybridizes under high stringency conditions to UBE2G1 (used as probe setpair that when added to (g) above can be used to generate a different4-gene biomarker that can distinguish patients with IBS from normalpatients).

In a second aspect, the present invention provides biomarkers,comprising or consisting of

(a) a first primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of TH1L,HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1,BLCAP, GPX1, HMGB1, and CALM3; and

(b) a second primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of TH1L,HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1,BLCAP, GPX1, HMGB1, and CALM3;

wherein the first primer pair and the second primer pair do notselectively amplify the same nucleic acid.

As is described in more detail below, the inventors have discovered thatthe biomarkers of the invention can be used, for example, as primers foramplification assays for diagnosing IBD and IBS. The biomarkers can beused, for example, to determine the expression levels in tissue mRNA forthe recited genes. The biomarkers of this second aspect of the inventionare especially preferred for use in RNA expression analysis from thegenes in a tissue of interest, such as blood samples (PBMCs orRBC-depleted whole blood).

The nucleic acid targets have been described in detail above, as havepolynucleotides in general. As used herein, “selectively amplifying”means that the primer pairs are complementary to their targets and canbe used to amplify a detectable portion of the nucleic acid target thatis distinguishable from amplification products due to non-specificamplification. In a preferred embodiment, the primers are fullycomplementary to their target.

As is well known in the art, polynucleotide primers can be used invarious assays (PCR, RT-PCR, RTQ-PCR, spPCR, qPCR, and allele-specificPCR, etc.) to amplify portions of a target to which the primers arecomplementary. Thus, a primer pair would include both a “forward” and a“reverse” primer, one complementary to the sense strand (ie: the strandshown in the sequences provided herein) and one complementary to an“antisense” strand (ie: a strand complementary to the strand shown inthe sequences provided herein), and designed to hybridize to the targetso as to be capable of generating a detectable amplification productfrom the target of interest when subjected to amplification conditions.The sequences of each of the target nucleic acids are provided herein,and thus, based on the teachings of the present specification, those ofskill in the art can design appropriate primer pairs complementary tothe target of interest (or complements thereof). In various embodiments,each member of the primer pair is a single stranded DNA polynucleotideat least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or more nucleotides in length that are fully complementary to thenucleic acid target. In various further embodiments, the detectableportion of the target nucleic acid that is amplified is at least 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or morenucleotides in length.

In various embodiments, the biomarker can comprise or consist of 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 primer pairs that selectivelyhybridizes under high stringency conditions to a nucleic acid selectedfrom the group consisting of TH1L, HIST1H2AC, TFE3, HIST1H2BK, UBE2G1,NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1, BLCAP, GPX1, HMGB1, and CALM3,wherein none of the 3-14 primer pairs selectively amplify the samenucleic acid. In a preferred embodiment, the primers are fullycomplementary to their target. Thus, as will be clear to those of skillin the art, the biomarkers may include further primer pairs that do notselectively amplify any of the recited human nucleic acids. Such furtherprimer pairs may include those consisting of polynucleotides thatselectively amplify other nucleic acids of interest, and may furtherinclude, for example, primer pairs to provide a standard ofamplification for comparison, etc.

In various embodiments of this second aspect, the biomarker consists of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 primer pairs.In various further embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more ofthe different primer pairs selectively amplify a detectable portion of anucleic acid selected from the group consisting of TH1L, HIST1H2AC,TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1, BLCAP,GPX1, HMGB1, and CALM3.

In one preferred embodiment of the second aspect of the invention, thebiomarker comprises or consists of at least 3, 4, or 5 primer pairs thatselectively amplify a detectable portion of a nucleic acid selected fromthe group consisting of TH1L, HIST1H2BK, UBE2G1, BLCAP, and CALM3. Asdisclosed in more detail below, such primer pairs can be used inpreferred embodiments of the methods of the invention for distinguishingnormal subjects from those subjects suffering from IBS. Examples of suchpreferred primer pairs are those that amplify a detectable portion ofthe nucleic acids provided in Table 17. Thus, in one preferred versionof this embodiment, the biomarker consists of between 3 and 35 differentnucleic acid primer pairs, wherein:

(a) a first primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:4 (TH1L), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3(BLCAP), and SEQ ID NO:6 (CALM3), wherein each primer in the firstprimer pair consists of 15 or more contiguous nucleotides of a nucleicacid selected from the group consisting of SEQ ID NO:4 (TH1L), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ IDNO:6 (CALM3), or a full complement thereof;

(b) a second primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:4 (TH1L), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3(BLCAP), and SEQ ID NO:6 (CALM3), wherein each primer in the secondprimer pair consists of 15 or more contiguous nucleotides of a nucleicacid selected from the group consisting of SEQ ID NO:4 (TH1L), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ IDNO:6 (CALM3), or a full complement thereof; and

(c) a third primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:4 (TH1L), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3(BLCAP), and SEQ ID NO:6 (CALM3), wherein each primer in the thirdprimer pair consists of 15 or more contiguous nucleotides of a nucleicacid selected from the group consisting of SEQ ID NO:4 (TH1L), SEQ IDNO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ IDNO:6 (CALM3), or a full complement thereof;

wherein each of the between 3 and 35 different primer pairs consists ofone or more primer pairs of 15 or more contiguous nucleotides, or fullcomplements thereof, for a single mRNA different from that of the otherprimer pairs, and wherein each of the different primer pairs isoptionally detectably labeled. In a further preferred version of thisembodiment, each primer in the first primer pair consists of 15 or morecontiguous nucleotides of BLCAP, or a full complement thereof; eachprimer in the second primer pair consists of 15 or more contiguousnucleotides of TH1L, or a full complement thereof; each primer in thethird primer pair consists of 15 or more contiguous nucleotides ofUBE2G1, or a full complement thereof; and each primer in a fourth primerpair consists of 15 or more contiguous nucleotides of HIST1H2BK, or afull complement thereof.

In another preferred embodiment of the second aspect of the invention,the biomarker comprises or consists of at least 5 or 6 primer pairs thatselectively amplify a detectable portion of a nucleic acid selected fromthe group consisting of RAP1A, BLCAP, UBE2G1, GPX1, CALM3, and NONO. Asdisclosed in more detail below, such primer pairs can be used inpreferred embodiments of the methods of the invention for distinguishingnormal subjects from those subjects suffering from IBD. Examples of suchpreferred primer pairs are those that amplify a detectable portion ofthe nucleic acids provided in Table 18. In one preferred version of thisembodiment, the biomarker consists of between 5 and 35 different nucleicacid primer pairs, wherein:

(a) a first primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO),wherein each primer in the first primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof;

(b) a second primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO)),wherein each primer in the second primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof;

(c) a third primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO),wherein each primer in the third primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof;

(d) a fourth primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO),wherein each primer in the fourth primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof; and

(e) a fifth primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO),wherein each primer in the fifth primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof;

wherein each of the between 5 and 35 different primer pairs consists ofone or more primer pairs of 15 or more contiguous nucleotides, or fullcomplements thereof, for a single mRNA different from that of the otherprimer pairs, and wherein each of the different primer pairs isoptionally detectably labeled. In a further embodiment, each primer inthe first primer pair consists of 15 or more contiguous nucleotides ofRAP1A, or a full complement thereof; each primer in the second primerpair consists of 15 or more contiguous nucleotides of BLCAP, or a fullcomplement thereof; each primer in the third primer pair consists of 15or more contiguous nucleotides of UBE2G1, or a full complement thereof;each primer in the fourth primer pair consists of 15 or more contiguousnucleotides of CALM3, or a full complement thereof; each primer in thefifth primer pair consists of 15 or more contiguous nucleotides of GPX1,or a full complement thereof; and each primer in a sixth primer pairconsists of 15 or more contiguous nucleotides of NONO, or a fullcomplement thereof.

In a further preferred embodiment of the second aspect of the invention,the biomarker comprises or consists of at least 6 or 7 primer pairs thatselectively amplify a detectable portion of a nucleic acid selected fromthe group consisting of RAP1A, BLCAP, UBE2G1, CALM3, GPX1, HIST1H2BK,and PPP2R5A. As disclosed in more detail below, such primer pairs can beused in preferred embodiments of the methods of the invention fordistinguishing those subjects suffering from IBS from those subjectssuffering from IBD. Examples of such preferred primer pairs are thosethat amplify a detectable portion of the nucleic acids provided in Table19. In one preferred embodiment, the biomarker consists of between 6 and35 different nucleic acid primer pairs, wherein:

(a) a first primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the first primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof;

(b) a second primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the second primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof;

(c) a third primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the third primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof;

(d) a fourth primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the fourth primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof;

(e) a fifth primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the fifth primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof; and

(f) a sixth primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ IDNO:7 and/or 8 (GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A),and SEQ ID NO:3 (BLCAP), wherein each primer in the sixth primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof;

wherein each of the between 6 and 35 different primer pairs consists ofone or more primer pairs of 15 or more contiguous nucleotides, or fullcomplements thereof, for a single mRNA different from that of the otherprimer pairs, and wherein each of the different primer pairs isoptionally detectably labeled. In another preferred embodiment, eachprimer in the first primer pair consists of 15 or more contiguousnucleotides of RAP1A, or a full complement thereof each primer in thesecond primer pair consists of 15 or more contiguous nucleotides ofUBE2G1, or a full complement thereof each primer in the third primerpair consists of 15 or more contiguous nucleotides of CALM3, or a fullcomplement thereof each primer in the fourth primer pair consists of 15or more contiguous nucleotides of GPX1, or a full complement thereofeach primer in the fifth primer pair consists of 15 or more contiguousnucleotides of HIST1H2BK, or a full complement thereof each primer inthe sixth primer pair consists of 15 or more contiguous nucleotides ofPPP2R5A, or a full complement thereof and wherein each primer in aseventh primer pair consists of 15 or more contiguous nucleotides ofBLCAP, or a full complement thereof.

In one specific embodiment, a biomarker according to this second aspectof the invention comprises or consists of a first primer pair thatselectively amplifies a detectable portion of BLCAP a second primer pairthat selectively amplifies a detectable portion of TH1L, a third primerpair that selectively amplifies a detectable portion of CALM3, and afourth primer pair that selectively amplifies a detectable portion ofHIST1H2BK. As disclosed in more detail below, the inventors havediscovered that such biomarkers can be used as probes to distinguishbetween normal and IBS patients.

In a second specific embodiment of this second aspect of the invention,a biomarker according to this second aspect of the invention comprisesor consists of a first primer pair that selectively amplifies adetectable portion of BLCAP, a second primer pair that selectivelyamplifies a detectable portion of TH1L, a third primer pair thatselectively amplifies a detectable portion of UBE2G1, and a fourthprimer pair that selectively amplifies a detectable portion ofHIST1H2BK. As disclosed in more detail below, the inventors havediscovered that such biomarkers can be used as probes to distinguishbetween normal and IBS patients.

In a third specific embodiment of this second aspect of the invention,the biomarker includes a first primer pair that selectively amplifies adetectable portion of RAP1A, a second primer pair that selectivelyamplifies a detectable portion of BLCAP, a third primer pair thatselectively amplifies a detectable portion of UBE2G1, and a fourthprimer pair that selectively amplifies a detectable portion of GPX1. Asdisclosed in more detail below, the inventors have discovered that suchbiomarkers can be used as probes to distinguish between normal and IBDpatients.

In a fourth specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a firstprimer pair that selectively amplifies a detectable portion of RAP1A, asecond primer pair that selectively amplifies a detectable portion ofBLCAP, a third primer pair that selectively amplifies a detectableportion of UBE2G1, a fourth primer pair that selectively amplifies adetectable portion of CALM3, and a fifth primer pair that selectivelyamplifies a detectable portion of GPX1. As disclosed in more detailbelow, the inventors have discovered that such biomarkers can be used asprobes to distinguish between normal and IBD patients

In a fifth specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a firstprimer pair that selectively amplifies a detectable portion of RAP1A, asecond primer pair that selectively amplifies a detectable portion ofBLCAP, a third primer pair that selectively amplifies a detectableportion of UBE2G1, a fourth primer pair that selectively amplifies adetectable portion of CALM3, a fifth primer pair that selectivelyamplifies a detectable portion of GPX1, and a sixth primer pair thatselectively amplifies a detectable portion of NONO. As disclosed in moredetail below, the inventors have discovered that such biomarkers can beused as probes to distinguish between normal and IBD patients.

In a sixth specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a primerpair that selectively amplifies a detectable portion of RAP1A, a secondprimer pair that selectively amplifies a detectable portion of BLCAP, athird primer pair that selectively amplifies a detectable portion ofUBE2G1, a fourth primer pair that selectively amplifies a detectableportion of CALM3, a fifth primer pair that selectively amplifies adetectable portion of GPX1, a sixth primer pair that selectivelyamplifies a detectable portion of HIST1H2BK, and a seventh primer pairthat selectively amplifies a detectable portion of PPP2RR5A. Asdisclosed in more detail below, the inventors have discovered that suchbiomarkers can be used as probes to distinguish normal and IBS patientsfrom IBD patients.

In a seventh specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a firstprimer pair that selectively amplifies a detectable portion of RAP1A, asecond primer pair that selectively amplifies a detectable portion ofUBE2G1, a third primer pair that selectively amplifies a detectableportion of CALM3, a fourth primer pair that selectively amplifies adetectable portion of GPX1, a fifth primer pair that selectivelyamplifies a detectable portion of HIST1H2BK, and a sixth primer pairthat selectively amplifies a detectable portion of PPP2R5A. As disclosedin more detail below, the inventors have discovered that such biomarkerscan be used as probes to distinguish between IBS patients and IBDpatients.

In an eighth specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a primerpair that selectively amplifies a detectable portion of RAP1A, a secondprimer pair that selectively amplifies a detectable portion of UBE2G1, athird primer pair that selectively amplifies a detectable portion ofCALM3, a fourth primer pair that selectively amplifies a detectableportion of GPX1, a fifth primer pair that selectively amplifies adetectable portion of HIST1H2BK, a sixth primer pair that selectivelyamplifies a detectable portion of PPP2R5A, and a seventh primer pairthat selectively amplifies a detectable portion of BLCAP. As disclosedin more detail below, the inventors have discovered that such biomarkerscan be used as probes to distinguish IBS patients from IBD patients.

In a ninth specific embodiment of this second aspect of the invention,the biomarker of this second aspect comprises or consists of a firstprimer pair that selectively amplifies a detectable portion of BLCAP, asecond primer pair that selectively amplifies a detectable portion ofCALM3, a third primer pair that selectively amplifies a detectableportion of GPX1, a fourth primer pair that selectively amplifies adetectable portion of TH1L, a fifth primer pair that selectivelyamplifies a detectable portion of RAP1A, and a sixth primer pair thatselectively amplifies a detectable portion of NONO. As disclosed in moredetail below, the inventors have discovered that such biomarkers can beused as probes to distinguish IBS and IBD patients from normal patients.

Thus, in various other embodiments of this second aspect of theinvention, the biomarker comprises or consists of:

(a) a first primer pair that selectively amplifies a detectable portionof RAP1A and a second primer pair that selectively amplifies adetectable portion of GPX1 (used as a primer pair set common tobiomarkers for diagnosing IBD from normal patients; IBD from normal andIBS; IBD from IBS; and IBD and IBS from normal);

(b) a first primer pair that selectively amplifies a detectable portionof UBE2G1 and a second primer pair that selectively amplifies adetectable portion of BLCAP (used as a primer pair set that can be addedto primer pair set (a) to generate a 4-gene biomarker specific fordiagnosing IBD from normal);

(c) a first primer pair that selectively amplifies a detectable portionof UBE2G1, a second primer pair that selectively amplifies a detectableportion of BLCAP, and a third primer pair that selectively amplifies adetectable portion of CALM3 (used as a primer pair trio that can beadded to primer pair (a) to generate a 5-gene biomarker for diagnosingIBD from normal);

(d) a first primer pair that selectively amplifies a detectable portionof NONO and a second primer pair that selectively amplifies a detectableportion of CALM3 (used as a primer pair set that, can be added to primerpair sets (a) and (b) to generate a 6-gene biomarker for diagnosing IBDfrom normal);

(e) a first primer pair that selectively amplifies a detectable portionof PPP2R5A and a second primer pair that selectively amplifies adetectable portion of HIST1H2BK (used as a primer pair set pair commonto biomarkers for diagnosing IBD from IBS patients). The primer pair set(e), when combined with primer pair sets (a), and (c) disclosed abovefor distinguishing IBD from normal, generates a biomarker for diagnosingnormal and IBS patients from IBD patients). The primer pair setcombination of (e), (a), and (c) can also be used as a biomarker todistinguish between IBS and IBD patients when the expression values ofthe individual genes are given different weights as discussed herein;

(f) a first primer pair that selectively amplifies a detectable portionof UBE2G1 and a second primer pair that selectively amplifies adetectable portion of CALM3 (used as a primer pair that can be added toprimer pair sets (a), and (e) described above, to generate a 6-genebiomarker that can be used to distinguish IBD from IBS patients);

(g) a first primer pair that selectively amplifies a detectable portionof BLCAP and a second primer pair that selectively amplifies adetectable portion of TH1L (used as primer pair set that when added to(a) and (d) above can be used to generate a 6-gene biomarker that candistinguish patients with IBS or IBD from normal patients;

h) a first primer pair that selectively amplifies a detectable portionof HIST1H2 KB and a second primer pair that selectively amplifies adetectable portion of CALM3 (used as primer pair set that when added to(g) above can be used to generate a 4-gene biomarker that candistinguish patients with IBS from normal patients; and

i) a first primer pair that selectively amplifies a detectable portionof HIST1H2 KB and a second primer pair that selectively amplifies adetectable portion of UBE2G1 (used as primer pair set that when added to(h) above can be used to generate a different 4-gene biomarker that candistinguish patients with IBS from normal patients.

The biomarkers of the first and second aspects of the invention can bestored frozen, in lyophilized form, or as a solution containing thedifferent probe sets or primer pairs. Such a solution can be made assuch, or the composition can be prepared at the time of hybridizing thepolynucleotides to target, as discussed below. Alternatively, thecompositions can be placed on a solid support, such as in a microarrayor microplate format.

In all of the above aspects and embodiments, the polynucleotides can belabeled with a detectable label. In a preferred embodiment, thedetectable labels for polynucleotides in different probe sets aredistinguishable from each other to, for example, facilitate differentialdetermination of their signals when conducting hybridization reactionsusing multiple probe sets. Methods for detecting the label include, butare not limited to spectroscopic, photochemical, biochemical,immunochemical, physical or chemical techniques. For example, usefuldetectable labels include but are not limited to radioactive labels suchas ³²P, ³H, and ¹⁴C; fluorescent dyes such as fluorescein isothiocyanate(FITC), rhodamine, lanthanide phosphors, and Texas red, ALEXIS™ (AbbottLabs), CY™ dyes (Amersham); electron-dense reagents such as gold;enzymes such as horseradish peroxidase, beta-galactosidase, luciferase,and alkaline phosphatase; colorimetric labels such as colloidal gold;magnetic labels such as those sold under the mark DYNABEADS™; biotin;dioxigenin; or haptens and proteins for which antisera or monoclonalantibodies are available. The label can be directly incorporated intothe polynucleotide, or it can be attached to a probe or antibody whichhybridizes or binds to the polynucleotide. The labels may be coupled tothe probes by any suitable means known to those of skill in the art. Invarious embodiments, the polynucleotides are labeled using nicktranslation, PCR, or random primer extension (see, e.g., Sambrook et al.supra).

In a third aspect, the present invention provides methods for diagnosingIBD and/or IBS comprising:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD or IBS under hybridizing conditions with2 or more probes sets, wherein at least a first probe set and a secondprobe set selectively hybridize under high stringency conditions to anucleic acid target selected from the group consisting of TH1L,HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A, PGRMC1,BLCAP, GPX1, HMGB1, and CALM3; wherein the first probe set and thesecond probe set do not selectively hybridize to the same nucleic acid;

(b) detecting formation of hybridization complexes between the 2 or moreprobe sets and nucleic acid targets in the nucleic acid sample, whereina number of such hybridization complexes provides a measure of geneexpression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD, IBS, orneither based on the gene expression of the nucleic acid targets.

The inventors have discovered that the methods of the invention can beused, for example, in diagnosing IBD and IBS. The specific genes, probesets, hybridizing conditions, probe types, polynucleotides, etc. are asdefined above for the first and/or second aspects of the invention.

The subject is any human subject that may be suffering from IBS or IBD.As discussed above, IBS is a chronic condition characterized byabdominal pain, constipation and/or diarrhea, and/or a change in bowelhabits while IBD patients may suffer from the same symptoms, as well asvomiting, hematochezia, weight loss, and/or weight gain; thus, forexample, subjects with one or more of these symptoms would be candidatesubjects for the methods of the invention.

As used herein, a “mRNA-derived nucleic acid sample” is a samplecontaining mRNA from the subject, or a cDNA (single or double stranded)generated from the mRNA obtained from the subject. The sample can befrom any suitable tissue source, including but not limited to bloodsamples, such as PBMCs or RBC-depleted whole blood.

In one embodiment, the mRNA sample is a human mRNA sample. It will beunderstood by those of skill in the art that the RNA sample does notrequire isolation of an individual or several individual species of RNAmolecules, as a complex sample mixture containing RNA to be tested canbe used, such as a cell or tissue sample analyzed by in situhybridization.

In a further embodiment, the probe sets comprise single strandedanti-sense polynucleotides of the nucleic acid compositions of theinvention. For example, in mRNA fluorescence in situ hybridization(FISH) (ie. FISH to detect messenger RNA), only an anti-sense probestrand hybridizes to the single stranded mRNA in the RNA sample, and inthat embodiment, the “sense” strand oligonucleotide can be used as anegative control.

Alternatively, the probe sets may comprise DNA probes. In either ofthese embodiments (anti-sense probes or cDNA probes), it is preferableto use controls or processes that direct hybridization to eithercytoplasmic mRNA or nuclear DNA. In the absence of directedhybridization, it is preferable to distinguish between hybridization tocytoplasmic RNA and hybridization to nuclear DNA.

Any method for evaluating the presence or absence of hybridizationproducts in the sample can be used, such as by Northern blottingmethods, in situ hybridization (for example, on blood smears),polymerase chain reaction (PCR) analysis, qPCR (quantitative PCR),RT-PCR (Real Time PCR), or array based methods.

In one embodiment, detection is performed by in situ hybridization(“ISH”). In situ hybridization assays are well known to those of skillin the art. Generally, in situ hybridization comprises the followingmajor steps (see, for example, U.S. Pat. No. 6,664,057): (1) fixation ofsample or nucleic acid sample to be analyzed; (2) pre-hybridizationtreatment of the sample or nucleic acid sample to increase accessibilityof the nucleic acid sample (within the sample in those embodiments) andto reduce nonspecific binding; (3) hybridization of the probe sets tothe nucleic acid sample; (4) post-hybridization washes to removepolynucleotides not bound in the hybridization; and (5) detection of thehybridized nucleic acid fragments. The reagent used in each of thesesteps and their conditions for use varies depending on the particularapplication. In a particularly preferred embodiment, ISH is conductedaccording to methods disclosed in U.S. Pat. Nos. 5,750,340 and/or6,022,689, incorporated by reference herein in their entirety.

In a typical in situ hybridization assay, cells are fixed to a solidsupport, typically a glass slide. The cells are typically denatured withheat or alkali and then contacted with a hybridization solution topermit annealing of labeled probes specific to the nucleic acid sequenceencoding the protein. The polynucleotides of the invention are typicallylabeled, as discussed above. In some applications it is necessary toblock the hybridization capacity of repetitive sequences. In this case,human genomic DNA or Cot-1 DNA is used to block non-specifichybridization.

When performing an in situ hybridization to cells fixed on a solidsupport, typically a glass slide, it is preferable to distinguishbetween hybridization to cytoplasmic RNA and hybridization to nuclearDNA. There are two major criteria for making this distinction: (1) copynumber differences between the types of targets (hundreds to thousandsof copies of RNA vs. two copies of DNA) which will normally createsignificant differences in signal intensities and (2) clearmorphological distinction between the cytoplasm (where hybridization toRNA targets would occur) and the nucleus will make signal locationunambiguous. Thus, when using double stranded DNA probes, it ispreferred that the method further comprises distinguishing the cytoplasmand nucleus in cells being analyzed within the bodily fluid sample. Suchdistinguishing can be accomplished by any means known in the art, suchas by using a nuclear stain such as Hoeschst 33342 or DAPI, whichdelineate the nuclear DNA in the cells being analyzed. In thisembodiment, it is preferred that the nuclear stain is distinguishablefrom the detectable probe. It is further preferred that the nuclearmembrane be maintained, i.e. that all the Hoeschst or DAPI stain bemaintained in the visible structure of the nucleus.

In a further embodiment, an array-based format can be used in which theprobe sets can be arrayed on a surface and the RNA sample is hybridizedto the polynucleotides on the surface. In this type of format, largenumbers of different hybridization reactions can be run essentially “inparallel.” This embodiment is particularly useful when there are manygenes whose expressions in one specimen are to be measured, or whenisolated nucleic acid from the specimen, but not the intact specimen, isavailable. This provides rapid, essentially simultaneous, evaluation ofa large number of gene expression assays. Methods of performinghybridization reactions in array based formats are also described in,for example, Pastinen (1997) Genome Res. 7:606-614; (1997) Jackson(1996) Nature Biotechnology 14:1685; Chee (1995) Science 274:610; WO96/17958. Methods for immobilizing the polynucleotides on the surfaceand derivatizing the surface are known in the art; see, for example,U.S. Pat. No. 6,664,057.

In each of the above aspects and embodiments, detection of hybridizationis typically accomplished through the use of a detectable label on thepolynucleotides in the probe sets, such as those described above; insome alternatives, the label can be on the target nucleic acids. Thelabel can be directly incorporated into the polynucleotide, or it can beattached to a probe or antibody which hybridizes or binds to thepolynucleotide. The labels may be coupled to the probes in a variety ofmeans known to those of skill in the art, as described above. The labelcan be detected by any suitable technique, including but not limited tospectroscopic, photochemical, biochemical, immunochemical, physical orchemical techniques, as discussed above.

The methods may comprise comparing gene expression of the nucleic acidtargets to a control. Any suitable control known in the art can be usedin the methods of the invention. For example, the expression level of agene known to be expressed at a relatively constant level in IBS, IBD,and normal patients can be used for comparison. Alternatively, theexpression level of the genes targeted by the probes can be analyzed innormal RNA samples equivalent to the test sample. Another embodiment isthe use of a standard concentration curve that gives absolute copynumbers of the mRNA of the gene being assayed; this might obviate theneed for a normalization control because the expression levels would begiven in terms of standard concentration units. Those of skill in theart will recognize that many such controls can be used in the methods ofthe invention.

The methods comprise diagnosing whether the subject is likely to haveIBD, IBS, or neither based on the gene expression of the nucleic acidtargets. As used herein, “likely to have” means a statisticallysignificant likelihood that the diagnosis is correct. In variousembodiments, the method results in an accurate diagnosis in at least 70%of cases; more preferably of at least 75%, 80%, 85%, 90%, or more of thecases.

The methods of the present invention may apply weights, derived byvarious means in the art, to the number of hybridization complexesformed for each nucleic acid target. Such means can be any suitable fordefining the classification rules for use of the biomarkers of theinvention in diagnosing IBD or IBS. Such classification rules can begenerated via any suitable means known in the art, including but notlimited to supervised or unsupervised classification techniques. In apreferred embodiment, classification rules are generated by use ofsupervised classification techniques. As used herein, “supervisedclassification” is a computer-implemented process through which eachmeasurement vector is assigned to a class according to a specifieddecision rule, where the possible classes have been defined on the basisof representative training samples of known identity. Examples of suchsupervised classification include, but are not limited to,classification trees, neural networks, k-nearest neighbor algorithms,linear discriminant analysis (LDA), quadratic discriminant analysis(QDA), and support vector machines.

In one non-limiting example, a weighted combination of the genes isarrived at by, for example, a supervised classification technique whichuses the expression data from all of the genes within individualpatients. The expression level of each gene in a patient is multipliedby the weighting factor for that gene, and those weighted values foreach gene's expression are summed for each individual patient, and,optionally, a separate coefficient specific for that comparison is addedto the sum which gives a final score. Each comparison set may result inits own specific set of gene weightings; as discussed further below, theIBD v Normal has different gene expression weightings than IBS v normal.Weightings can also have either a positive-sign or a negative-sign. Notall patients in one classification will have the same Gene 1 up, Gene 2down, etc. (See examples below).

In various embodiments of this third aspect of the invention, the two ormore probe sets comprise or consist of at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 probe sets, and wherein none of the 3-14 probe setsselectively hybridize to the same nucleic acid. These embodiments ofprobe sets are further discussed in the first and second aspects of theinvention; all other embodiments of the probe sets and polynucleotidesof the first and second aspect can be used in the methods of theinvention.

In one preferred embodiment of the third aspect of the invention, atleast 3, 4, or 5 of the probe sets selectively hybridize under highstringency conditions to a nucleic acid selected from the groupconsisting of TH1L, HIST1H2BK, UBE2G1, BLCAP, and CALM3. As disclosed inmore detail below, this method can be used in preferred embodiments ofthe methods of the invention for distinguishing normal subjects fromthose subjects suffering from IBS. Examples of such preferred probe setsfor use in the methods of the invention are provided in Table 17. In onepreferred version of this embodiment, is a method for diagnosingirritable bowel syndrome (IBS), comprising:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBS under hybridizing conditions with atleast 3 nucleotide probes, wherein a first nucleotide probe, a secondnucleotide probe, and a third nucleotide probe each consist of 15 ormore contiguous nucleotides of a nucleic acid target selected from thegroup consisting of SEQ ID NO:4 (TH1L), SEQ ID NO:9 (HIST1H2BK), SEQ IDNO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), and SEQ ID NO:6 (CALM3), or a fullcomplement thereof, wherein each of the first, second, and thirdnucleotide probes consist of 15 or more contiguous nucleotides of adifferent nucleic acid target;

(b) detecting formation of hybridization complexes between the probesand the nucleic acid targets in the nucleic acid sample, wherein anumber of such hybridization complexes provides a measure of geneexpression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBS based on themeasure of gene expression of the nucleic acid targets. In a furtherpreferred version of this embodiment, the nucleic acid targets compriseBLCAP, TH1L, UBE2G1, and HIST1H2BK. In a further preferred version ofthis embodiment, the subject suffers from one or more of abdominal pain,constipation, diarrhea, and a change in bowel habits. In another versionof this embodiment, the mRNA-derived nucleic acid sample is obtainedfrom peripheral blood mononuclear cells red blood cell-depleted wholeblood. In a further version of this embodiment, the method furthercomprises analyzing gene expression of the nucleic acid targets byapplying a weight to the number of hybridization complexes formed foreach nucleic acid target.

In another preferred embodiment of the third aspect of the invention, atleast 5 or 6 of the probe sets selectively hybridize under highstringency conditions to a nucleic acid selected from the groupconsisting of RAP1A, BLCAP, UBE2G1, GPX1, CALM3, and NONO. As disclosedin more detail below, such probe sets can be used in preferredembodiments of the methods of the invention for distinguishing normalsubjects from those subjects suffering from IBD. Examples of suchpreferred probe sets for use in the methods of the invention areprovided in Table 18. In one preferred version of this embodiment, themethod comprises:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD under hybridizing conditions with atleast 5 nucleotide probes, wherein a first nucleotide probe, a secondnucleotide probe, a third nucleotide probe, a fourth nucleotide probe,and a fifth nucleotide probe each consist of 15 or more contiguousnucleotides of a nucleic acid target selected from the group consistingof SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ IDNO:13 (NONO), or a full complement thereof, wherein each of the first,second, third, fourth, and fifth nucleotide probes consist of 15 or morecontiguous nucleotides of a different nucleic acid target;

(b) detecting formation of hybridization complexes between thenucleotide probes and the nucleic acid targets in the nucleic acidsample, wherein a number of such hybridization complexes provides ameasure of gene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD based on themeasure of gene expression of the nucleic acid targets. In a furtherpreferred embodiment, the nucleic acid targets comprise RAP1A, BLCAP,UBE2G1, CALM3, GPX1, and NONO. In another embodiment, the subject may besuffering from one or more of abdominal pain, constipation, diarrhea, achange in bowel habits, vomiting, hematochezia, and weight change. In afurther embodiment, the mRNA-derived nucleic acid sample is obtainedfrom peripheral blood mononuclear cells or red blood cell-depleted wholeblood. In a further embodiment, the method further comprises analyzinggene expression of the nucleic acid targets by applying a weight to thenumber of hybridization complexes formed for each nucleic acid target.

In a further preferred embodiment of the third aspect of the invention,at least 6 or 7 of the probe sets selectively hybridize under highstringency conditions to a nucleic acid selected from the groupconsisting of RAP1A, BLCAP, UBE2G1, CALM3, GPX1, HIST1H2BK, and PPP2R5A.As disclosed in more detail below, such probe sets can be used inpreferred embodiments of the methods of the invention for distinguishingthose subjects suffering from IBS from those subjects suffering fromIBD. Examples of such preferred probe sets for use in the methods of theinvention are provided in Table 19. In one preferred version, of thisembodiment, a method for differentiating between inflammatory boweldisease (IBD) and irritable bowel syndrome (IBS) in a subject,comprises:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD under hybridizing conditions with atleast 6 nucleotide probes, wherein a first nucleotide probe, a secondnucleotide probe, a third nucleotide probe, a fourth nucleotide probe, afifth nucleotide probe, and a sixth nucleotide probe each consist of 15or more contiguous nucleotides of a nucleic acid target selected fromthe group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), SEQ ID NO:9(HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3 (BLCAP), or a fullcomplement thereof; wherein each of the first, second, third, fourth,fifth, and sixth nucleotide probes consist of 15 or more contiguousnucleotides of a different nucleic acid target;

(b) detecting formation of hybridization complexes between thenucleotide probes and the nucleic acid targets in the nucleic acidsample, wherein a number of such hybridization complexes provides ameasure of gene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD or IBS based onthe measure of gene expression of the nucleic acid targets. In a furtherpreferred embodiment, the nucleic acid targets comprise RAP1A, UBE2G1,CALM3, GPX1, HIST1H2BK, PPP2R5A, and BLCAP. In another embodiment, thesubject suffers from one or more of abdominal pain, constipation,diarrhea, a change in bowel habits, vomiting, hematochezia, and weightchange. In a further embodiment, the mRNA-derived nucleic acid sample isobtained from peripheral blood mononuclear cells or red bloodcell-depleted whole blood. In another embodiment, the method furthercomprises analyzing gene expression of the nucleic acid targets byapplying a weight to the number of hybridization complexes formed foreach nucleic acid target.

In one specific embodiment of this third aspect of the invention, themethods comprise use of a first probe set that selectively hybridizesunder high stringency conditions to BLCAP, a second probe set thatselectively hybridizes under high stringency conditions to TH1L, a thirdprobe set that selectively hybridizes under high stringency conditionsto CALM3, and a fourth probe set that selectively hybridizes under highstringency conditions to HIST1H2BK. As disclosed in more detail below,the inventors have discovered that such methods can be used todistinguish between normal and IBS patients.

In a second specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to BLCAP, a second probe setthat selectively hybridizes under high stringency conditions to TH1L, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, and a fourth probe set that selectively hybridizesunder high stringency conditions to HIST1H2BK. As disclosed in moredetail below, the inventors have discovered that such methods can beused to distinguish between normal and IBS patients.

In a third specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1. As disclosed in more detailbelow, the inventors have discovered that such methods can be used asprobes to distinguish between normal and IBD patients.

In a fourth specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, and a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1. Asdisclosed in more detail below, the inventors have discovered that suchmethods can distinguish between normal and IBD patients.

In a fifth specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1, and asixth probe set that selectively hybridizes under high stringencyconditions to NONO. As disclosed in more detail below, the inventorshave discovered that such methods can distinguish between normal and IBDpatients.

In a sixth specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to BLCAP, athird probe set that selectively hybridizes under high stringencyconditions to UBE2G1, a fourth probe set that selectively hybridizesunder high stringency conditions to CALM3, a fifth probe set thatselectively hybridizes under high stringency conditions to GPX1, a sixthprobe set that selectively hybridizes under high stringency conditionsto HIST1H2BK, and a seventh probe set that selectively hybridizes underhigh stringency conditions to PPP2RR5A. As disclosed in more detailbelow, the inventors have discovered that such methods can distinguishnormal and IBS patients from IBD patients.

In a seventh specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to UBE2G1,a third probe set that selectively hybridizes under high stringencyconditions to CALM3, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1, a fifth probe set thatselectively hybridizes under high stringency conditions to HIST1H2BK1,and a sixth probe set that selectively hybridizes under high stringencyconditions to PPP2R5A. As disclosed in more detail below, the inventorshave discovered that such methods can be used as probes to distinguishIBS patients from IBD patients.

In an eighth specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to RAP1A, a second probe setthat selectively hybridizes under high stringency conditions to UBE2G1,a third probe set that selectively hybridizes under high stringencyconditions to CALM3, a fourth probe set that selectively hybridizesunder high stringency conditions to GPX1, a fifth probe set thatselectively hybridizes under high stringency conditions to HIST1H2BK, asixth probe set that selectively hybridizes under high stringencyconditions to PPP2R5A, and a seventh probe set that selectivelyhybridizes under high stringency conditions to BLCAP. As disclosed inmore detail below, the inventors have discovered that such methods canbe used as probes to distinguish IBS patients from IBD patients.

In a ninth specific embodiment of this third aspect of the invention,the methods comprise use of a first probe set that selectivelyhybridizes under high stringency conditions to BLCAP, a second probe setthat selectively hybridizes under high stringency conditions to CALM3, athird probe set that selectively hybridizes under high stringencyconditions to GPX1, a fourth probe set that selectively hybridizes underhigh stringency conditions to TH1L, a fifth probe set that selectivelyhybridizes under high stringency conditions to RAP1A, and a sixth probeset that selectively hybridizes under high stringency conditions toNONO. As disclosed in more detail below, the inventors have discoveredthat such methods can be used as probes to distinguish IBS and IBDpatients from normal patients.

In a fourth aspect, the present invention provides methods method fordiagnosing IBD and/or IBS comprising:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD or IBS under amplifying conditions with2 or more primer pairs, wherein at least a first primer pair and asecond primer pair are capable of selectively amplifying a detectableportion of a nucleic acid target selected from the group consisting ofTH1L, HIST1H2AC, TFE3, HIST1H2BK, UBE2G1, NONO, PCBP1, RAP1A, PPP2R5A,PGRMC1, BLCAP, GPX1, HMGB1, and CALM3; wherein the first primer pair andthe second primer pair do not selectively amplify the same nucleic acid;

(b) detecting amplification products generated by amplification ofnucleic acid targets in the nucleic acid sample by the two or moreprimer pairs, wherein the amplification products provide a measure ofgene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD, IBS, orneither based on the amplification of the nucleic acid targets.

Definitions of primer pairs as used above apply to this aspect of theinvention, as well as all other common terms. All embodiments disclosedabove for the other aspects of the invention are also suitable for thisfourth aspect.

In these methods, amplification of target nucleic acids using the primerpairs is used instead of hybridization to detect gene expressionproducts. Any suitable amplification technique can be used, includingbut not limited to PCR, RT-PCT, qPCR, spPCR, etc. Suitable amplificationconditions can be determined by those of skill in the art based on theparticular primer pair design and other factors, based on the teachingsherein. In various embodiments, the two or more primer pairs comprise atleast 3-14 primer pairs, wherein none of the 3-14 primer pairsselectively amplify the same nucleic acid.

In one preferred embodiment of the fourth aspect of the invention, atleast 3, 4, or 5 of the primer pairs selectively amplify a detectableportion of a nucleic acid selected from the group consisting of TH1L,HIST1H2BK, UBE2G1, BLCAP, and CALM3. As disclosed in more detail below,such primer pairs can be used in preferred embodiments of the methods ofthe invention for distinguishing normal subjects from those subjectssuffering from IBS. Examples of such preferred primer pairs are thosethat amplify a detectable portion of the nucleic acids provided in Table17. In one preferred version of this embodiment, a method for diagnosingirritable bowel syndrome (IBS) comprises:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBS under amplifying conditions with atleast three primer pairs, wherein a first primer pair, a second primerpair, and a third primer pair selectively amplify a different nucleicacid target selected from the group consisting of SEQ ID NO:4 (TH1L),SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ ID NO:3 (BLCAP), andSEQ ID NO:6 (CALM3), or a full complement thereof, wherein each primerin each primer pair consists of at least 15 contiguous nucleotides ofits respective nucleic acid target;

(b) detecting amplification products generated by amplification ofnucleic acid targets in the nucleic acid sample by the at least threeprimer pairs, wherein a number of such amplification products provides ameasure of gene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBS based on themeasure of gene expression of the nucleic acid targets. In a furtherversion of this embodiment, the nucleic acid targets comprise BLCAP,TH1L, UBE2G1, and HIST1H2BK. In another version, the subject suffersfrom one or more of abdominal pain, constipation, diarrhea, and a changein bowel habits. The mRNA-derived nucleic acid sample may be obtainedfrom peripheral blood mononuclear cells red blood cell-depleted wholeblood. The method may further comprise analyzing gene expression of thenucleic acid targets by applying a weight to the number of amplificationproducts formed for each nucleic acid target.

In another preferred embodiment of the fourth aspect of the invention,at least 5 or 6 of the primer pairs selectively amplify a detectableportion of a nucleic acid selected from the group consisting of RAP1A,BLCAP, UBE2G1, GPX1, CALM3, and NONO. As disclosed in more detail below,such primer pairs can be used in preferred embodiments of the methods ofthe invention for distinguishing normal subjects from those subjectssuffering from IBD. Examples of such preferred primer pairs are thosethat amplify a detectable portion of the nucleic acids provided in Table18. In a preferred embodiment, the method for IBD diagnosis comprises

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD under amplifying conditions with atleast five primer pairs, wherein a first primer pair, a second primerpair, a third primer, a fourth primer pair, and a fifth primer pair eachselectively amplify a different nucleic acid target selected from thegroup consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP),SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1),and SEQ ID NO:13 (NONO), or a full complement thereof, wherein eachprimer in each primer pair consists of at least 15 contiguousnucleotides of its respective nucleic acid target;

(b) detecting amplification products generated by amplification ofnucleic acid targets in the nucleic acid sample by the at least fiveprimer pairs, wherein a number of such amplification products provides ameasure of gene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD based on themeasure of gene expression of the nucleic acid targets. In a furtherpreferred embodiment, the nucleic acid targets comprise RAP1A, BLCAP,UBE2G1, CALM3, GPX1, and NONO. In another embodiment, the subjectsuffers from one or more of abdominal pain, constipation, diarrhea, achange in bowel habits, vomiting, hematochezia, and weight change. In afurther embodiment, the mRNA-derived nucleic acid sample is obtainedfrom peripheral blood mononuclear cells or red blood cell-depleted wholeblood. In another embodiment, the method further comprises analyzinggene expression of the nucleic acid targets by applying a weight to thenumber of amplification products formed for each nucleic acid target.

In a further preferred embodiment of the fourth aspect of the invention,at least 6 or 7 of the primer pairs selectively amplify a detectableportion of a nucleic acid selected from the group consisting of RAP1A,BLCAP, UBE2G1, CALM3, GPX1, HIST1H2BK, and PPP2R5A. As disclosed in moredetail below, such primer pairs can be used in preferred embodiments ofthe methods of the invention for distinguishing those subjects sufferingfrom IBS from those subjects suffering from IBD. Examples of suchpreferred primer pairs are those that amplify a detectable portion ofthe nucleic acids provided in Table 19. In one preferred version, methodfor differentiating between inflammatory bowel disease (IBD) andirritable bowel syndrome (IBS) in a subject comprises:

(a) contacting a mRNA-derived nucleic acid sample obtained from asubject suspected of having IBD under amplifying conditions with atleast six primer pairs, wherein a first primer pair, a second primerpair, a third primer, a fourth primer pair, a fifth primer pair, and asixth primer pair each selectively amplify a different nucleic acidtarget selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8(GPX1), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:10 (PPP2R5A), and SEQ ID NO:3(BLCAP), or a full complement thereof, wherein each primer in eachprimer pair consists of at least 15 contiguous nucleotides of itsrespective nucleic acid target;

(b) detecting amplification products generated by amplification ofnucleic acid targets in the nucleic acid sample by the at least sixprimer pairs, wherein a number of such amplification products provides ameasure of gene expression of the nucleic acid targets; and

(c) diagnosing whether the subject is likely to have IBD or IBS based onthe measure of gene expression of the nucleic acid targets. In apreferred embodiment, the nucleic acid targets comprise RAP1A, UBE2G1,CALM3, GPX1, HIST1H2BK, PPP2R5A, and BLCAP. In a further embodiment, thesubject suffers from one or more of abdominal pain, constipation,diarrhea, a change in bowel habits, vomiting, hematochezia, and weightchange. In another embodiment, the mRNA-derived nucleic acid sample isobtained from peripheral blood mononuclear cells or red bloodcell-depleted whole blood. In a further embodiment, the method furthercomprises analyzing gene expression of the nucleic acid targets byapplying a weight to the number of amplification products formed foreach nucleic acid target.

In one specific embodiment of this fourth aspect of the invention, themethods comprise use of a first primer pair that selectively amplifies adetectable portion of BLCAP, a second primer pair that selectivelyamplifies a detectable portion of TH1L, a third primer pair thatselectively amplifies a detectable portion of CALM3, and a fourth primerpair that selectively amplifies a detectable portion of HIST1H2BK. Asdisclosed in more detail below, the inventors have discovered that suchmethods can distinguish between normal and IBS patients.

In a second specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of BLCAP, a second primer pair thatselectively amplifies a detectable portion of TH1L, a third primer pairthat selectively amplifies a detectable portion of UBE2G1, and a fourthprimer pair that selectively amplifies a detectable portion ofHIST1H2BK. As disclosed in more detail below, the inventors havediscovered that such methods can distinguish between normal and IBSpatients.

In a third specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of RAP1A, a second primer pair thatselectively amplifies a detectable portion of BLCAP, a third primer pairthat selectively amplifies a detectable portion of UBE2G1, and a fourthprimer pair that selectively amplifies a detectable portion of GPX1. Asdisclosed in more detail below, the inventors have discovered that suchmethods can be used as probes to distinguish between normal and IBDpatients.

In a fourth specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of RAP1A, a second primer pair thatselectively amplifies a detectable portion of BLCAP, a third primer pairthat selectively amplifies a detectable portion of UBE2G1, a fourthprimer pair that selectively amplifies a detectable portion of CALM3,and a fifth primer pair selectively amplifies a detectable portion ofGPX1. As disclosed in more detail below, the inventors have discoveredthat such methods can distinguish between normal and IBD patients.

In a fifth specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of RAP1A, a second primer pair thatselectively amplifies a detectable portion of BLCAP, a third primer pairthat selectively amplifies a detectable portion of UBE2G1, a fourthprimer pair that selectively amplifies a detectable portion of CALM3, afifth primer pair selectively amplifies a detectable portion of GPX1,and a sixth primer pair selectively amplifies a detectable portion ofNONO. As disclosed in more detail below, the inventors have discoveredthat such methods can distinguish between normal and IBD patients.

In a sixth specific embodiment of this fourth aspect of the invention,the methods comprise use of a primer pair that selectively amplifies adetectable portion of RAP1A, a second primer pair that selectivelyamplifies a detectable portion of BLCAP, a third primer pair thatselectively amplifies a detectable portion of UBE2G1, a fourth primerpair that selectively amplifies a detectable portion of CALM3, a fifthprimer pair that selectively amplifies a detectable portion of GPX1, asixth primer pair that selectively amplifies a detectable portion ofHIST1H2BK; and a seventh primer pair selectively that amplifies adetectable portion of PPP2RR5A. As disclosed in more detail below, theinventors have discovered that such methods can distinguish normal andIBS patients from IBD patients.

In a seventh specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of RAP1A, a second primer pair thatselectively amplifies a detectable portion of UBE2G1, a third primerpair that selectively amplifies a detectable portion of CALM3, a fourthprimer pair that selectively amplifies a detectable portion of GPX1, afifth primer pair selectively amplifies a detectable portion ofHIST1H2BK, and a sixth primer pair selectively amplifies a detectableportion of PPP2R5A. As disclosed in more detail below, the inventorshave discovered that such methods can be used to distinguish between IBSpatients and IBD patients

In an eighth specific embodiment of this fourth aspect of the invention,the methods comprise use of a primer pair that selectively amplifies adetectable portion of RAP1A, a second primer pair that selectivelyamplifies a detectable portion of UBE2G1, a third primer pair thatselectively amplifies a detectable portion of CALM3, a fourth primerpair that selectively amplifies a detectable portion of GPX1, a fifthprimer pair that selectively amplifies a detectable portion ofHIST1H2BK, a sixth primer pair that selectively amplifies a detectableportion of PPP2R5A, and a seventh primer pair selectively that amplifiesa detectable portion of BLCAP. As disclosed in more detail below, theinventors have discovered that such methods can be used as probes todistinguish IBS patients from IBD patients.

In a ninth specific embodiment of this fourth aspect of the invention,the methods comprise use of a first primer pair that selectivelyamplifies a detectable portion of BLCAP, a second primer pair thatselectively amplifies a detectable portion of CALM3, a third primer pairthat selectively amplifies a detectable portion of GPX1, a fourth primerpair that selectively amplifies a detectable portion of TH1L, a fifthprimer pair selectively amplifies a detectable portion of RAP1A, and asixth primer pair selectively amplifies a detectable portion of NONO. Asdisclosed in more detail below, the inventors have discovered that suchmethods can be used as probes to distinguish IBS and IBD patients fromnormal patients.

In various embodiments, the methods may further comprise comparingamplification products to a control.

In a further embodiment of all of the methods of the invention, themethods are automated, and appropriate software is used to conduct someor all stages of the method.

In a further aspect, the present invention provides kits for use in themethods of the invention, comprising the biomarkers and/or primer pairsets of the invention and instructions for their use. In a preferredembodiment, the polynucleotides are detectably labeled, most preferablywhere the detectable labels on each polynucleotide in a given probe setor primer pair are the same, and differ from the detectable labels onthe polynucleotides in other probe sets or primer pairs, as disclosedabove. In a further preferred embodiment, the probes/primer pairs areprovided in solution, most preferably in a hybridization oramplification buffer to be used in the methods of the invention. Infurther embodiments, the kit also comprises wash solutions,pre-hybridization solutions, amplification reagents, software forautomation of the methods, etc.

EXAMPLE 1

In an effort to identify gene expression profiles that coulddiscriminate between whole blood samples collected from IBS, IBD, andnormal patients, and thus provide the basis for a minimally invasivediagnostic test, we employed a proprietary data mining program toanalyze publicly available data collected from Crohn's Disease (CD) andUlcerative Colitis (UC) patients (Burczynski et al., MolecularClassification of Crohn's Disease and Ulcerative Colitis Patients UsingTranscriptional Profiles in Peripheral Blood Mononuclear Cells, Journalof Molecular Diagnostics 8(1):51-61, February 2006), hereinafterreferred to as the “Burczynski data.”

The Burczynski data consisted of a set of individual expression levelfeatures, each feature being a quantitative fluorescent signal derivedfrom a single microarray spot. As detailed in Burczynski et al (2006),the signals were generated by hybridizing fluorescently-labeled RNA froma single patient to all of the spots on a single DNA-basedoligonucleotide microarray. From these data, we identified molecularsignatures, comprised of sets of expression level features, thateffectively differentiated between IBD patients and unaffected normalcontrol subjects. Expression levels of the genes represented by thosearray features were then measured in a prospectively ascertained sampleof patients (the ‘pilot study’, described below). The Burczynskidiscovery dataset consisted of 127 separate Affymetrix microarrayhybridization experiments on RNA from 26 Ulcerative Colitis patients, 59Crohn's Disease patients, and 42 normal controls.

We employed our proprietary data mining program to analyze the publiclyavailable Burczynski data. We randomly divided the patients in theBurczynski dataset for purposes of our analysis into 2 approximatelyequal groups: a training set for biomarker set discovery and a separatenon-overlapping test set for assessment of biomarkers discovered fromthe training set. The training set consisted of 30 CD patients, 13 UCpatients, and 21 normal controls. The test set consisted of 29 CDpatients, 13 UC patients, and 21 controls. The CD and UC patients weredefined as ‘affected’, and normal controls were defined as ‘unaffected’.

Our proprietary data mining program was then used to perform agenetic-algorithm search of expression level data for combinationsacross the affected and unaffected patient sets. The number of featuresconstituting a marker set combination was fixed at 4. The Burczynskidata set contained 22,283 expression level features; the number of4-wise combinations of features in that data set are:(!22,283/(!4*!22,279)=10,269,905,646,716,170.

The proprietary data mining program was run 3 separate times on theBurczynski dataset using three specific sets of parameters. (1) Oneparameter set used the training and test sets defined above withadditional settings that gave computational results weighted towardshigher sensitivity (to minimize false negatives), (2) the second set wassimilarly weighted towards higher specificity (to minimize falsepositives), and (3) the third set used random cross-validation(‘bootstrap’) with no weighting towards either specificity orsensitivity. Each 4-feature combination analyzed was assigned a scorethat characterized its accuracy in discriminating between the affectedand unaffected groups. The score for each combination of expressionfeatures ranges from 1.00 for completely accurate to 0.00 for completelyinaccurate.

For the set weighted towards higher sensitivity (set 1), the top-scoring4-feature sets were obtained such that a combination's score on thetraining set was greater than 0.995, or the combination's score on thetest set was greater than 0.92. For the set weighted towards higherspecificity (set 2), the top-scoring 4-feature sets were obtained suchthat a combination's score on the training set was greater than 0.9975,or the combination's score on the test set was greater than 0.92. Forthe bootstrap result set with equal weighting between sensitivity andspecificity (set 3), the top-scoring 4-feature sets were obtained suchthat a combination's score on the training set was greater than 0.995(ie: approximately 99.5% accuracy).

Significance of the marker sets was assessed empirically by randomiterative relabeling. The affected and unaffected statuses of patientswere randomly re-assigned, and the proprietary data mining program wasthen run to determine the top marker solutions for the randomly labeledset. This was repeated to obtain 100,000 marker sets. In the randomlyrelabeled sets, the training set scores reached a maximum of 0.927; 95%of solutions (the empirical p=0.05 level) scored at or below 0.882, and99% of solutions the empirical p=0.01 level) scored at or below 0.893.The test set scores in relabeled solutions reached a maximum of 0.915;95% of solutions (the empirical p=0.05 level) scored at or below 0.753,and 99% of solutions (the empirical p=0.01 level) scored at or below0.809.

A total of sixteen sets, each comprised of 4 features (in the Burczynskimicroarray data, some genes are represented by more than 1 feature andsome features hybridize to more than 1 gene), was obtained using acombination of thresholds: the score on the training set was greaterthan 0.9975 and/or the score on the test set was greater than 0.92.

Table 2 contains the combinations of genes that were identified from thegene expression profile of peripheral blood mononuclear cells thateffectively differentiate between IBD patients and unaffected normalcontrol subjects.

TABLE 2 Top 16 4-gene combinations Combination Gene 1 Gene 2 Gene 3 Gene4 1 HIST1H2BK NONO PPP2R5A BLCAP 2 TH1L PPP2R5A PGRMC1 BLCAP 3 TH1LUBE2G1 PPP2R5A BLCAP 4 TH1L PPP2R5A BLCAP HMGB1 5 HIST1H2BK RAP1APPP2R5A BLCAP 6 NONO PPP2R5A BLCAP GPX1 7 HIST1H2AC RAP1A PPP2R5A HMGB18 TH1L TFE3 UBE2G1 BLCAP 9 PCBP1 RAP1A PPP2R5A PGRMC1 10 NONO UBE2G1PPP2R5A BLCAP 11 TH1L HIST1H2AC PPP2R5A BLCAP 12 UBE2G1 PPP2R5A BLCAPGPX1 13 HIST1H2AC PPP2R5A BLCAP GPX1 14 TH1L PPP2R5A BLCAP CALM3 15 TH1LRAP1A PPP2R5A CALM3 16 TH1L HIST1H2BK PPP2R5A BLCAP

Fourteen individual expression array features constitute those 16 sets.The feature sets and their memberships are indicated in Table 3 below.Each feature represents the expression level of a transcript from asingle gene; the HUGO gene names for each feature are indicated. Theaverage fold-difference in expression between the IBD and normal groupsis also shown, computed by dividing the average expression level in IBDpatients by the average expression level in Normal patients. Afold-difference greater than 1 indicates the gene has higher expressionin IBD patients compared to normal patients, while a fold-differenceless than 1 indicates the gene has lower expression in IBD patientscompared to normal patients. The row labeled “freq” shows how many timesthat microarray feature occurs in the top 16 marker sets.

TABLE 3 Gene frequency in top 16 combinations HGNC name PPP2R5A BLCAPTH1L UBE2G1 RAP1A HIST1H2AC HIST1H2BK IBD/NML 0.76 0.72 0.70 0.76 1.602.13 1.94 fold diff Feature # 20569 21724 2313 13649 20394 7839 12991freq 15 13 8 4 4 3 3 14 genes in X X X 16 sets X X X X X X X X X X X X XX X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X HGNCname GPX1 NONO PGRMC1 HMGB1 CALM3 TFE3 PCBP1 IBD/NML 1.80 0.78 1.70 0.721.42 1.41 1.31 fold diff Feature # 22020 12337 21635 22076 22134 1044214167 freq 3 3 2 2 2 1 1 14 genes in X 16 sets X X X X X X X X X X X X X

A gene list (Table 4) was derived from an analysis (Table 3) of theunique set of genes in these combinations.

TABLE 4 Gene List gene name affy # Hs.ID gene description TH1L220607_x_at Hs.517148 TH1-like (Drosophila) HIST1H2AC 215071_s_atHs.484950 histone 1, H2ac TFE3 212457_at Hs.274184 Transcription factorbinding to IGHM enhancer 3 NONO 210470_x_at Hs.533282 Non-POU domaincontaining, octamer-binding HIST1H2BK 209806_at Hs.437275 Histone 1,H2bk UBE2G1 209141_at Hs.462035 Ubiquitin-conjugating enzyme E2G 1 (UBC7homolog, C. elegans) PCBP1 208620_at Hs.2853 Poly(rC) binding protein 1RAP1A 202362_at Hs.190334 RAP1A, member of RAS oncogene family PPP2R5A202187_s_at Hs.497684 Protein phosphatase 2, regulatory subunit B (B56),alpha isoform PGRMC1 201121_s_at Hs.90061 Progesterone receptor membranecomponent 1 BLCAP 201032_at Hs.472651 Bladder cancer associated proteinGPX1 200736_s_at Hs.76686 Glutathione peroxidase 1 HMGB1 200680_x_atHs.434102 High-mobility group box 1 CALM3 200622_x_at Hs.515487Calmodulin 3 (phosphorylase kinase, delta)

An example of the diagnostic performance of the discovery biomarker setson the analyzed dataset is shown in FIG. 1. A logistic regression wasperformed with the patient's affected/unaffected status against thequantitative levels of the four features from a discovery marker set.The training set patient data were used to perform the regressionanalysis. The resulting regression coefficients were then used tocalculate a weighted sum of the expression levels of the four featuresin the test set patients. The resulting weighted-sum regression scorefor each training set patient was then plotted, with IBD patients havingopen symbols and Normal patients having filled symbols. In the exampleshown in the graph below, a regression-score cutoff of approximately2.75 assigns an ‘affected’ test result to 40 patients with IBD and to 1Normal patient. An ‘unaffected’ result is assigned to 2 patients withIBD and 20 Normal patients. Using standard calculations for medicaldiagnostics, this gives Sensitivity=40/42=0.952,Specificity=20/21=0.952, Positive Predictive Value=40/41=0.976, andNegative Predictive Value=20/22=0.909.

EXAMPLE 2

Eight of the genes listed in Table 4 that occurred frequently in the topsets shown in Table 3 were evaluated, along with Actin B as a controlgene, in a subsequent study on a total of 482 RBC (red bloodcell)-depleted whole blood samples from: 98 normal controls, 91Ulcerative Colitis patients, 98 Crohn's Disease patients, 98 IBSpatients and 97 patients with non-gastrointestinal inflammatory disease.Samples were obtained from 7 clinical sites at various geographiclocations within the U.S.A. In the analysis there was no distinctionmade between Crohn's and UC.

The RNA expression levels of eight genes from among the 14 features inthe top discovery marker sets were measured by quantitative real-timePCR in the RBC-depleted whole blood of a prospectively ascertainedsample of affected patients and unaffected controls. An additional ninthgene was measured to serve as an internal reference standard acrosspatients. Five cohorts of patients had RNA assayed:

-   -   98 Normal    -   98 Crohn's Disease    -   91 Ulcerative Colitis    -   98 Irritable Bowel Syndrome    -   97 Non-Gastrointestinal Inflammatory Disease

The 9 genes (including 1 control) assayed for RNA expression levels inthe 5 cohorts of patients were: (Human Genome Organization (HUGO) GeneNomenclature Committee names are given)

-   -   RAP1A    -   BLCAP    -   PPP2R5A    -   UBE2G1    -   GPX1    -   TH1-L    -   CALM3    -   HIST1H2BK    -   ACTB (the internal reference gene)

Whole blood samples and clinical information were obtained from allpatients. Each IBD patient was diagnosed by a board-certifiedgastroenterologist; IBD diagnoses were confirmed by endoscopy. Each IBSpatient was diagnosed by a board-certified gastroenterologist using RomeI criteria. All protocols were IRB approved; informed consent wasobtained and peripheral blood samples and clinical data were collectedfrom all patients. RNA was isolated, cDNA was synthesized, andquantitative real-time PCR for each gene was performed on an AppliedBiosystems 7300 Real-Time PCR System. Expression levels were output asCt (cycle or crossing threshold). DeltaCts of each gene for each patientwere computed to normalize gene expression levels of the 8 marker genesto the intra-subject reference gene (ACTB also referred to as β-Actin).DeltaCt=Ct[marker gene]−Ct[ACTB]. The DeltaCts were then used foranalysis of diagnostic classification performance.

2A. IBD vs. Normal 5-Gene Combination

An example of biomarker performance is shown in FIG. 2, the boxplots ofthe logistic regression scores based on expression levels of 5 of the 8genes whose combination differentiates between normal and IBD patients(RAP1A, BLCAP, UBE2G1, CALM3, and GPX1). The classification matrix anddiagnostic accuracy estimates of the combination for IBD v Normal areshown below the graph (Tables 5a-b).

TABLE 5a IBD vs Normal (5-gene) Classification Matrix IBD Normal test >0 162 25 test ≦ 0 22 71 Fisher exact OR = 20.57, p < 2 × 10⁻¹⁶

TABLE 5b Performance Measures study prevalence Accuracy 83.2%Sensitivity 88.0% Specificity 74.0% Positive Predictive Value (PPV)86.6% Negative Predictive Value (NPV) 76.3%2B. IBD vs. Normal 4-Gene CombinationAn initial logistic regression model was built using data for all eightgenes. The model was trimmed by eliminating genes not statisticallysignificantly contributing to the model. The result of this analysis isa four-gene model incorporating: RAP1A, BLCAP, UBE2G1, and GPX1. Theaccuracy of this model in distinguishing IBD samples from normal samplesis 80.4%, as shown in Table 6b, below, along with the other performancemeasures of this 4-gene combination.

TABLE 6a IBD vs Normal (4-gene) Classification Matrix IBD Normal test >0 159 30 test ≦ 0 25 66 Fisher exact OR = 13.81, p < 2 × 10⁻¹⁶

TABLE 6b Performance Measures study prevalence Acc 80.4% Sens 86.4% Spec68.8% PPV 84.1% NPV 72.5%2C. Normal & IBS vs. IBD 7 Gene Combination

An example of biomarker performance is shown in FIG. 3, boxplots of thelogistic regression scores based on expression levels of 7 of the 8genes whose combination differentiates between normal/IBS and IBDpatients (RAP1A, BLCAP, UBE2G1, CALM3, GPX1, HIST1H2BK, AND PPP2R5A).The classification matrix and diagnostic accuracy estimates (Tables7a-b) of the combination for IBD v Normal/IBS are shown below the graph.

TABLE 7a IBS&Normal vs IBD (7-gene) Classification Matrix IBD IBS +Normal test > 0 141 44 test ≦ 0 43 150 Fisher exact OR = 11.09, p < 2 ×10⁻¹⁶

TABLE 7b Performance measures study prevalence Acc 77.0% Sens 76.6% Spec77.3% PPV 76.2% NPV 77.7%2D. IBS vs. Normal 4-Gene Combination

An initial logistic regression model was built using data for all eightgenes. The model was trimmed by eliminating genes not statisticallysignificantly contributing to the model. The result of this analysis isa four-gene model incorporating: BLCAP, TH1L, CALMS, and HIST1H2BK. Theaccuracy of this model in distinguishing IBS samples from normal samplesis 84.7%.

The biomarker performance is in FIG. 4, boxplots of the logisticregression scores based on expression levels of 4 of the 8 genes whosecombination differentiates between Normal and IBS patients. Theclassification matrix and diagnostic accuracy estimates of thecombination for Normal versus IBS are shown in Tables 8a-b, below thegraph.

TABLE 8a IBS vs Normal (4-gene) Classification Matrix IBS Normal test >0 83 16 test ≦ 0 15 82 Fisher exact OR = 27.62, p < 2 × 10⁻¹⁶

TABLE 8b Performance measures study prevalence Acc 84.2% Sens 84.7% Spec83.7% PPV 83.8% NPV 84.5%2E. IBD vs. IBS 6-Gene Combination

As above, an initial logistic regression model was built using data forall eight genes. The model was trimmed by eliminating genes notstatistically significantly contributing to the model. The result ofthis analysis is a six-gene model incorporating: RAP1A, UBE2G1, CALM3,GPX1, HISTH2BK, and PPP2R5A. The accuracy of this model indistinguishing IBS samples from IBD samples is 83.0%.

TABLE 9a IBD vs IBS differentiation IBD IBS test > 0 163 27 test ≦ 0 2171 Fisher exact OR = 20.09, p < 2 × 10⁻¹⁶

TABLE 9b Performance measures study prevalence Acc 83.0% Sens 88.6% Spec72.4% PPV 85.8% NPV 77.2%Summary: The ability of the expression levels of these genes, asmeasured by RT-PCR and normalized by Actin B expression, to individuallydistinguish between IBD and normal controls; IBD from IBS; IBD fromNormal & IBS; and IBS from Normal are shown in Table 10, (below). The pvalues for significant differences in expression levels for each gene,between each pair of cohorts are shown. To the right of each p-value,the experimental values (the ΔCt) are shown as “+” (or “−”) to indicatea higher (or lower) ΔCt for one member of the subject pair relative tothe other (see legend to Table 10). The ΔCt is inversely proportional tothe expression level.

TABLE 10 Significantly different expression levels of individual genes pvalues (Wilcoxon rank sum test) Probe IBD vs normal IBD vs IBS IBD vsnorm&IBS IBS vs normal RAP1A 4.22 × 10⁻¹⁶ + 5.24 × 10⁻⁵ +  1.62 ×10⁻¹³ + 6.25 × 10⁻⁷ + BLCAP 3.62 × 10⁻¹⁵ + 0.78 na 3.59 × 10⁻⁶ +  9.45 ×10⁻¹⁶ + TH1L 6.78 × 10⁻⁷  + 0.69 na  1.0 × 10⁻³ + 7.86 × 10⁻⁶ + UBE2G18.34 × 10⁻¹² + 0.003 − 0.018 +  <2.2 × 10⁻¹⁶ + CALM3 3.57 × 10⁻¹⁰ + 0.24na 5.53 × 10⁻⁶ + 9.79 × 10⁻⁷ + GPX1 0.099 na 0.007 − 7.59 × 10⁻³ − 0.34na HIST1H2BK 0.069 na 1.02 × 10⁻⁶ − 0.061 na 3.85 × 10⁻⁹ + PPP2R5A 5.13× 10⁻⁶  + 7.73 × 10⁻³ − 0.25  na  7.25 × 10⁻¹² + Legend: IBD vsnormal: + = higher ΔCt in IBD relative to normal; − = lower ΔCt in IBDrelative to normal IBD vs IBS: + = higher ΔCt in IBD relative to IBS; −= lower ΔCt in IBD IBD vs normal&IBS: + = higher ΔCt in IBD relative tonormal&IBS; − = lower ΔCt in IBD IBS vs normal: + = higher ΔCt in IBSrelative to normal; − = lower ΔCt in IBS All: na = not applicablebecause p-value is not significant In all cases, the ΔCt is inverselyproportional to expression level. Therefore a lower ΔCt represents ahigher level of gene expression and a higher ΔCt represents a lowerlevel of gene expression.

While all eight genes were found to be statistically significantlyassociated with IBD or IBS, the individual genes are not highly accuratein discriminating the various subgroups. We investigated if combinationsof genes selected from the eight might enable clinically useful markeraccuracies.

For each of the data subsets: IBD vs. normal control, IBS vs. normalcontrol, IBS and Normal vs. IBD, and IBD vs. IBS, we evaluated theaccuracy of gene combinations using logistic regression. One skilled inthe art will understand that given a set of measurements, such as thegene expression values for a particular set of genes, and given thesemeasurements across a particular set of samples, such as a group of IBDsamples and a group of ‘normal’ samples, there are many techniques forderiving from that data a ‘set of rules’ for classifying a sample as,e.g., IBD or normal. Those skilled in the art will understand that analgorithm, including a weighting for each gene expression level, willfollow from the logistic regression analysis, according to one method ofthe body of knowledge known as ‘supervised learning’, which is asub-field of ‘machine learning’, which itself can be consideredsub-field of ‘data mining’. Supervised learning encompasses techniquesfor deriving algorithms, or rules, from data. One skilled in the artwill understand that there are no clear boundaries between a standardstatistical approach, and a ‘supervised learning’ approach, and that theclassification formulas presented below could be considered as beingderived from a supervised learning approach, but could also be termed astandard statistical approach.

The boxplots in the examples above are a visual display of thedistribution of the individual scores within each cohort for theweightings that were optimized for a given comparison. The threshold forthe classification score is zero in all cases. For IBD v Normal, IBD isgreater than zero and Normal is less than zero; the IBS and Othercategories are included as informational, but they were not used indetermining the gene expression weightings. Similarly for IBD v Normal &IBS, IBD is greater than zero and Normal & IBS are less than zero, andthe Other category was not used in deriving the weightings. And the samefor IBS v Normal: IBS is greater than zero and Normal is less than zero,and the IBD and Other categories weren't used to derive expressionweightings. Thus, the specific ups and downs of the expression levels ofindividual genes in the marker set do matter in the classifier, but notin a direct always-up or always-down manner. What matters is whether thesum of the weighted expression values is greater than or less than zero.A specific gene may have increased expression in one correctlyclassified patient, and that same gene may have a decreased expressionin another correctly classified patient if the score is “compensated” byappropriately weighted changes in the expression of other genes in themarker set.

By way of non-limiting example, in one specific measurement, thefollowing gene weightings were applied:

IBS vs Normal

4 gene BLCAP, TH1L, CALM3, HIST1H2BK

If(−16.6856+11.2898(BLCAP)−4.9722(TH1L)−3.7663(CALM3)+2.5060(HIST1H2BK))>0

Then IBS Else Normal

For IBS versus Normal and given the gene weightings above and athreshold of zero with IBS greater than zero and Normal less than orequal to zero, a hypothetical patient with expression levels ofBLCAP=2.0, TH1L=1.0, CALM3=1.0, and HIST1H2BK=2.0 would have a weightedscore of(−16.6856+11.2898(2.0)−4.9722(1.0)−3.7663(1.0)+2.5060(2.0)=2.1675,resulting in a classification of IBS.

Other non-limiting examples of such weightings are as follows:

IBS & Normal vs IBD

7-gene RAP1A, BLCAP, UBE2G1, CALM3, GPX1, HIST1H2BK, PPP2R5A

If(−0.1602+4.487(RAP1A)+2.5746(BLCAP)−2.8938(UBE2G1)+3.8415(CALM3)−2.3682(GPX1)−1.5623(HIST1H2BK)−1.4927(PPP2RR5A))>0

Then IBD Else IBS or Normal IBD vs Normal

4-gene RAP1A, BLCAP, UBE2G1, GPX1

If(−10.8729+3.3467(RAP1A)+5.1866(BLCAP)−3.4559(UBE2G1)+3.237(CALM3)−3.225(GPX1))>0 Then IBD Else Normal IBD vs Normal

5-gene RAP1A, BLCAP, UBE2G1, CALM3, GPX1:

Formula:(−10.7716+ΔCt_(RAP1A)*3.2485+ΔCt_(BLCAP)*5.4360−ΔCt_(UBE2G1)*3.4723+ΔCt_(CALM3)*3.3511−ΔCt_(GPX1)*3.4642)

If(−10.7716+ΔCt_(RAP1A)*3.2485+ΔCt_(BLCAP)*5.4360−ΔCt_(UBE2G1)*3.4723+ΔCt_(CALM3)*3.3511−ΔCt_(GPX1)*3.4642)>0

Then IBD Else Normal IBS vs IBD

6-gene RAP1A, UBE2G1, CALM3, GPX1, HIST1H2BK, PPP2R5A:

Formula:(16.6895+ΔCt_(RAP1A)*5.1550−ΔCt_(UBE2G1)*2.5462+ΔCt_(CALM3)*4.8674−ΔCt_(GPX1)*2.3979−ΔCt_(HIST1H2BK)*2.8183−ΔCt_(PPP2R5A)*2.9778)

If16.6895+ΔCt_(RAP1A)*5.1550−ΔCt_(UBE2G1)*2.5462+ΔCt_(CALM3)*4.8674−ΔCt_(GPX1)*2.3979−ΔCt_(HIST12BK)*2.8183−ΔCt_(PPP2R5A)*2.9778>0.0

Then IBD ELSE NORMAL

One exemplary classification rule derived from logistic regressionanalysis of the Burczynski et al (2006) data for classifying IBD vs.Normal using the 5-gene IBD vs Normal biomarker would be:

IF

−1.1704−0.2815*RAP1A+0.7027*BLCAP+0.3143*UBE2G1+0.1089*CALM3−0.4782*GPX1is less than 0

THEN IBD ELSE NORMAL

actual predicted IBD Normal IBD 82 1 Normal 3 41

Accuracy 96.9% Sensitivity 96.5% Specificity 97.6% PPV 98.8% NPV 93.2%FIG. 5 shows the box plots of the regression scores based on expressionlevels of 5 genes whose combination differentiates IBD from normal(RAP1A, BLCAP, UBE2G1, CALM3, and GPX1).

The weightings disclosed above are optimal examples; as will be clear tothose of skill in the art, the weightings can vary widely based on, forexample, the use of a different classifier to generate the weightings,or the use of modified weightings that may provide lower accuracy butimproved specificity (or some other change to the assay of interest to auser).

EXAMPLE 3

In a separate study, ten of the genes listed in Table 4 that occurredmost frequently in the top sets shown in Table 3 were evaluated, alongwith Actin B as a control gene, in a subsequent study on a total of 482RBC-depleted whole blood samples from: 98 normal controls, 91 UlcerativeColitis patients, 98 Crohn's Disease patients, 98 IBS patients and 97patients with non-gastrointestinal inflammatory disease. Samples wereobtained from 7 clinical sites at various geographic locations withinthe U.S.A.; in the analysis there was no distinction made betweenCrohn's and UC.

The RNA expression levels of ten genes from among the 15 features in thetop discovery marker sets were measured by quantitative real-time PCR inthe RBC-depleted whole blood of a prospectively ascertained sample ofaffected patients and unaffected controls. An additional eleventh genewas measured to serve as an internal reference standard across patients.Five cohorts of patients had RNA assayed:

-   -   98 Normal    -   98 Crohn's Disease    -   91 Ulcerative Colitis    -   98 Irritable Bowel Syndrome    -   97 Non-Gastrointestinal Inflammatory Disease

The 11 genes (including 1 control) assayed for RNA expression levels inthe 5 cohorts of patients were: (Human Genome Organization (HUGO) GeneNomenclature Committee names are given)

-   -   RAP1A    -   BLCAP    -   PPP2R5A    -   UBE2G1    -   GPX1    -   TH1-L    -   CALM3    -   HIST1H2BK    -   NONO    -   HMGB1    -   ACTB (the internal reference gene)

Whole blood samples and clinical information were obtained from allpatients. Each IBD patient was diagnosed by a board-certifiedgastroenterologist; IBD diagnoses were confirmed by endoscopy. Each IBSpatient was diagnosed by a board-certified gastroenterologist using RomeI criteria. All protocols were IRB approved; informed consent wasobtained and peripheral blood samples and clinical data were collectedfrom all patients. Expression data were obtained from peripheral wholeblood samples (with no mononuclear enrichment) by isolating total mRNAs,synthesizing cDNAs, and performing real-time quantitative PCR on anApplied Biosystems 7300 Real-Time PCR System. Expression levels wereoutput as Ct (cycle or crossing threshold). DeltaCts of each gene foreach patient were computed to normalize gene expression levels of the 10marker genes to the intra-subject reference gene (ACTB also referred toas β-Actin). DeltaCt=Ct[marker gene]−Ct[ACTB]. The DeltaCts were thenused for analysis of diagnostic classification performance.

3A IBD vs Normal (6-Gene)

An optimal scoring algorithm for classification of patients as IBD ornormal was derived based on 6 of the 10 tested genes (RAP1A, BLCAP,UBE2G1, CALM3, GPX1, NONO). The classification matrix and diagnosticperformance measures for this biomarker are given in Table 11a and 11b,respectively.

TABLE 11a IBD vs Normal (6-gene) Classification Matrix ClinicalDiagnosis IBD Normal Biomarker IBD 168 25 Classification Normal 21 73Fisher's exact Odds Ratio (2-sided) = 23.0, p < 2 × 10−16

TABLE 11b Diagnostic Performance Measures accuracy* 84% sensitivity 89%specificity 75% positive predictive value* 87% negative predictivevalue* 78% AUC-ROC 0.91 *result based on study prevalences.The NPV for an adjusted 25% prior probability of IBD (a rule-outscenario) is 95%. The PPV for an adjusted 75% prior probability of IBD(a rule-in scenario) is 91%.

3B. IBS vs Normal (4-Gene)

An optimal scoring algorithm for classification of patients as IBS ornormal was derived based on 4 of the 10 tested genes (BLCAP, UBE2G1,TH1L, HIST1H2BK). The classification of patients by clinical diagnosisand test result is given in Table 12a. The diagnostic performance of theclassification is summarized in Table 12b.

TABLE 12a IBS vs Normal (4-gene) Classification Matrix ClinicalDiagnosis IBS Normal Biomarker IBS 87 21 Classification Normal 11 77Fisher's exact Odds Ratio (2-sided) = 28.3, p < 2 × 10⁻¹⁶

TABLE 12b Diagnostic Performance Measures accuracy* 84% sensitivity 89%specificity 79% positive predictive value* 81% negative predictivevalue* 88% AUC-ROC 0.92 *result based on study prevalences

The NPV for an adjusted 25% prior probability of IBS (a rule-outscenario) is 96%. The PPV for an adjusted 75% prior probability of IBS(a rule-in scenario) is 93%.

3C. IBS vs IBD (7 Gene)

An optimal scoring algorithm for classification of patients as IBS orIBD was derived based on 7 of the 10 tested genes (RAP1A, UBE2G1, CALM3,GPX1, HIST1H2BK, PPP2R5A, BLCAP). The classification of patients byclinical diagnosis and test result is given in Table 13a. The diagnosticperformance of the classification is summarized in Table 13b.

TABLE 13a IBS vs IBD (7-gene) Classification Matrix IBD IBS test > 0 16326 test ≦ 0 21 72 Fisher's exact OR = 21.14, p < 2 × 10⁻¹⁶

TABLE 13b Clinical Performance Measures study prevalence Acc 83.3% Sens88.6% Spec 73.5% PPV 86.2% NPV 77.4%

The PPV for an adjusted 75% prior probability of IBD (a rule-in IBDscenario) is 90.9%. The NPV for an adjusted 25% prior probability of IBD(a rule-out IBD scenario) is 95.1%.

3D (IBX vs Normal)

An optimal scoring algorithm for classification of patients as eitherIBS or IBD vs normal was derived based on 6 of the 10 tested genes(6-gene BLCAP, CALM3, GPX1, TH1L, RAP1A, NONO IBX vs Normal). Theclassification of patients by clinical diagnosis and test result isgiven in Table 14a. The diagnostic performance of the classification issummarized in Table 14b.

TABLE 14a IBX vs Normal (6-gene) Classification Matrix IBX norm test > 0269 45 test ≦ 0 18 53 Fisher's exact OR = 17.4, p < 2 × 10⁻¹⁶

TABLE 14b Clinical Performance Measures at study prevalence Acc 83.6%Sens 93.7% Spec 54.1% PPV 85.7% NPV 74.6%

3E Shared Biology of Genes in Biomarkers

Expression levels of the 10 candidate biomarker genes in Example 3 wereassayed on each patient specimen and normalized to a within-patientreference gene. Optimal scoring algorithms for classification ofpatients as IBD versus normal and IBS versus normal were derivedseparately using the expression levels of the 10 genes assayed in thesepilot study patients. The optimal gene set for each test and each set'sperformance is indicated in Table 15. The classification results hadodds ratios of 23.0 and 28.3, respectively, with both p-values <2×10⁻¹⁶.Two genes were common to both diagnostic marker sets. The genesidentified as diagnostic for the comparisons reveal an overlap betweenIBD and IBS patients. This overlap of highly statistically significantbiomarkers suggests a shared biology between the two disease states. Thecommonality of the two genes could indicate that genes have a causativerole or that they are part of a common response mechanism.

TABLE 15 Highly significant expression biomarker sets in IBD and IBS*Cohort sub set odds ratio and p-value IBD vs Normal IBS vs Normal OR =23.0, p = 2 × 10⁻¹⁶ OR = 28.3, p = 2 × 10⁻¹⁶ (Fisher's 2-sided exact)(Fisher's 2-sided exact) genes in marker sets Expression level*Expression level* BLCAP + + UBE2G1 + + TH1L + CALM3 + HIST1H2BK + GPX1n.a. NONO + RAP1A + *univariate ΔCt (inversely proportional toexpression level) difference relative to Normal: “+” increased; “−”decreased; “+/−” no difference; n.a. = not significantly differentSummary: The ability of the expression levels of these 10 genes, asmeasured by RT-PCR and normalized by Actin B expression, to individuallydistinguish between IBD and normal controls; IBD from IBS; IBS fromNormal; and IBS&IBD from normal are shown in Table 16 (below). The pvalues for significant differences in expression levels for each gene,between each pair of subjects are shown. To the right of each p-value,the experimental values (the ΔCt) are shown as “+” (or “−”) to indicatea higher (or lower) ΔCt for one member of the subject pair relative tothe other (see legend to Table 16). The ΔCt is inversely proportional tothe expression level.

TABLE 16 Significantly different expression levels of individual genesProbe IBD vs normal IBD vs IBS IBS vs normal IBS&IBD vs normal RAP1A4.22 × 10⁻¹⁶ + 5.24 × 10⁻⁵ + 6.25 × 10⁻⁷ + 8.24 × 10⁻¹⁵ + BLCAP 3.62 ×10⁻¹⁵ + 0.78 na  9.45 × 10⁻¹⁶ + <2.2 × 10⁻¹⁶ + TH1L 6.78 × 10⁻⁷  + 0.69na 7.86 × 10⁻⁶ + 8.89 × 10⁻⁸  + UBE2G1 8.34 × 10⁻¹² + 0.003 −  <2.2 ×10⁻¹⁶ + <2.2 × 10⁻¹⁶ + CALM3 3.57 × 10⁻¹⁰ + 0.24 na 9.79 × 10⁻⁷ + 1.19 ×10⁻¹⁰ + GPX1 0.099 na 0.007 − 0.34 na 0.45 na HIST1H2BK 0.069 na 1.02 ×10⁻⁶ − 3.85 × 10⁻⁹ + 1.89 × 10⁻⁴  + PPP2R5A 5.13 × 10⁻⁶  + 7.73 × 10⁻³ − 7.25 × 10⁻¹² + 1.38 × 10⁻⁹  + NONO 6.70 × 10⁻⁵  + 0.053 na 0.08 na 4.50× 10⁻⁴  + HMGB1 4.61 × 10⁻¹⁰ + 0.47 na 0.16 na 4.24 × 10⁻¹⁰ + Legend:IBD vs normal: + = higher ΔCt in IBD relative to normal; − = lower ΔCtin IBD relative to normal IBD vs IBS: + = higher ΔCt in IBD relative toIBS; − = lower ΔCt in IBD IBS vs normal: + = higher ΔCt in IBS relativeto normal; − = lower ΔCt in IBS IBS&IBD vs normal: + = higher ΔCt inIBS&IBD relative to normal; − = lower ΔCt in IBS&IBD All: na = notapplicable because p-value is not significant In all cases, the ΔCt isinversely proportional to expression level. Therefore a lower ΔCtrepresents a higher level of gene expression and a higher ΔCt representsa lower level of gene expression.

While all ten genes were found to be statistically significantlyassociated with IBD or IBS, the individual genes are not highly accuratein discriminating the various subgroups. We investigated whethercombinations of genes selected from the ten might enable clinicallyuseful marker accuracies.

For each of the data subsets: IBD vs. normal control, IBS vs. normalcontrol, IBS vs. IBD, and IBD& IBS vs. normal, we evaluated the accuracyof gene combinations using logistic regression. One skilled in the artwill understand that given a set of measurements, such as the geneexpression values for a particular set of genes, and given thesemeasurements across a particular set of samples, such as a group of IBDsamples and a group of ‘normal’ samples, there are many techniques forderiving from that data a ‘set of rules’ for classifying a sample as egIBD or normal. Those skilled in the art will understand that analgorithm, including a weighting for each gene expression level, willfollow from the logistic regression analysis, according to one method ofthe body of knowledge known as ‘supervised learning’, which is asub-field of ‘machine learning’, which itself can be considered asub-field of ‘data mining’. Supervised learning encompasses techniquesfor deriving algorithms, or rules, from data. One skilled in the artwill understand that there are no clear boundaries between a standardstatistical approach, and a ‘supervised learning’ approach, and that theclassification formulas presented below could be considered as beingderived from a supervised learning approach, but could also be termed astandard statistical approach.

The threshold for the classification score is zero in all cases. For IBDv Normal, IBD is greater than zero and Normal is less than zero; the IBSand Other were not used in determining the gene expression weightings.Similarly for IBS vs. Normal, IBS is greater than zero and Normal isless than zero, and the Other categories were not used in deriving theweightings. In the case of IBD vs. IBS, IBD is greater than zero and IBSis less than zero and the Normal and Other categories were not used toderive expression weightings. Finally, in the case of IBD and IBS vs.normal, a score greater than zero indicates either IBD or IBS; a scoreless than zero indicates Normal, and the Other category was not used toderive the weightings. Thus, the specific ups and downs of theexpression levels of individual genes in the marker set do matter in theclassifier, but not in a direct always-up or always-down manner withrespect to the disease or non-disease status patient. Of more importanceis whether the sum of the weighted expression values is greater than orless than zero. A specific gene may have increased expression in onecorrectly classified patient, and that same gene may have a decreasedexpression in another correctly classified patient if the score is“compensated” by appropriately weighted changes in the expression ofother genes in the marker set.

The following exemplary gene weightings were applied:

IBD vs Normal 6-Gene RAP1A, BLCAP, UBE2G1, CALM3, GPX1, NONO:

IBD Diagnostic Index:=(−16.5312+4.7721[dCt_(BLCAP)]−3.3249[dCt_(GPX1)]₊3.6521[dCt_(RAP1A)]−3.0221[dCt_(UBE2G1)]+3.0669[dCt_(CALM3)]+0.8405[dCt_(NONO)])

If(−16.5312+4.7721[dCt_(BLCAP)]−3.3249[dCt_(GPX1)]₊3.6521[dCt_(RAP1A)]−3.0221[dCt_(UBE2G1)]+3.0669[dCt_(CALM3)]+0.8405[dCt_(NONO)])>0

Then IBD Else Normal IBS vs Normal 4-Gene BLCAP, TH1L, UBE2G1,HIST1H2BK:

Formula:=−22.1323+5.4337[dCt_(BLCAP)]+3.3187[dCt_(UBE2G1)]−4.1747[dCt_(TH1L)]+1.6902[dCt_(HIST1H2BK)]

If[−22.1323+5.4337[dCt_(BLCAP)]+3.3187[dCt_(UBE2G1)]−4.1747[dCt_(TH1L)]+1.6902[dCt_(HIST1H2BK)]>0

Then IBS Else Normal IBD vs IBS 7-Gene RAP1A, UBE2G1, CALM3, GPX1,HIST1H2BK, PPP2R5A, BLCAP:

Formula:(17.6564+ΔCt_(RAP1A)*5.7988−ΔCt_(UBE2G1)*5.6479+ΔCt_(CALM3)*3.4257−ΔCt_(GPX1)*2.5535−ΔCt_(HIST1H2BK)*2.1366−ΔCt_(PPP2R5A)*2.4503+ΔCt_(BLCAP)*3.0325)

IF(17.6564+ΔCt_(RAP1A)*5.7988−ΔCt_(UBE2G1)*5.6479+ΔCt_(CALM3)*3.4257−ΔCt_(GPX1)*2.5535−ΔCt_(HIST1H2BK)*2.1366−ΔCt_(PPP2R5A)*2.4503+ΔCt_(BLCAP)*3.0325)>0

Then IBD Else IBS IBX vs Normal 6-Gene BLCAP, CALM3, GPX1, TH1L, RAP1A,NONO:

Formula:(−13.5528+ΔCt_(BLCAP)*4.9346+ΔCt_(CALM3)*2.8244−ΔCt_(GPX1)*1.8043−ΔCt_(TH1L)*2.8452+ΔCt_(RAP1A)*1.2203+ΔCt_(NONO)*1.04)

IF(−13.5528+ΔCt_(BLCAP)*4.9346+ΔCt_(CALM3)*2.8244−ΔCt_(GPX1)*1.8043−ΔCt_(TH1L)*2.8452+ΔCt_(RAP1A)*1.2203+ΔCt_(NONO)*1.04)>0

Then IBD or IBS Else Normal

We subsequently analyzed the biomarker performance based on expressionlevels of combinations of 3 of the 5 genes tested above (Examples 2D and3B), whose combination differentiates between normal and IBS patients(BLCAP, TH1L, CALM3, HIST1H2BK, and UBE2G1). The classification matrixand diagnostic accuracy estimates of the combination for IBS v Normal,as well as exemplary gene weightings, are shown in Table 17.

TABLE 17 2 × 2 table combination IBS Normal fisher's accuracysensitivity specificity PPV NPV BLCAP test > 0 81 19 OR = 19.39 81.6%82.7% 80.6% 81.0% 82.3% TH1L test ? 0 17 79 p < 2 × 10⁻¹⁶ CALM3equation: −10.0504 + 8.4473* ΔCt_(BLCAP) − 5.4222* ΔCt_(TH1L) + 2.4570 *ΔCt_(CALM3) BLCAP test > 0 88 20 OR = 33.38 84.7% 89.8% 79.6% 81.5%88.6% TH1L test ? 0 10 78 p < 2 × 10⁻¹⁶ UBE2G1 equation: −16.4275 +5.0435* ΔCt_(BLCAP) − 4.4467* ΔCt_(TH1L) + 4.1683 * ΔCt_(UBE2G1) BLCAPtest > 0 82 16 OR = 27.62 84.2% 84.5% 83.8% 83.7% 84.7% TH1L test ? 0 1583 p < 2 × 10⁻¹⁶ HIST1H2BK equation: −17.5471 + 8.2126* ΔCt_(BLCAP) −4.2986* ΔCt_(TH1L) + 2.1835* ΔCt_(HIST1H2BK) BLCAP test > 0 83 23 OR =17.68 80.6% 84.7% 76.5% 78.3% 83.3% CALM3 test ? 0 15 75 p < 2 × 10⁻¹⁶UBE2G1 equation: −21.741 + 0.6081*ΔCt_(BLCAP) − 0.5865*ΔCt_(CALM3) +3.933*ΔCt_(UBE2G1) BLCAP test > 0 83 25 OR = 15.86 79.6% 84.7% 74.5%76.9% 83.0% CALM3 test ? 0 15 73 p < 2 × 10⁻¹⁶ HIST1H2BK equation:−24.6537 + 4.4848* ΔCt_(BLCAP) − 2.1685* ΔCt_(CALM3) + 2.9207*ΔCt_(HIST1H2BK) BLCAP test > 0 83 27 OR = 14.30 78.6% 84.7% 72.4% 75.5%82.6% UBE2G1 test ? 0 15 71 p = 3.0 × 10⁻¹⁶ HIST1H2BK equation:−27.6040 + 1.2774* ΔCt_(BLCAP) + 2.8181* ΔCt_(UBE2G1) + 1.7602*ΔCt_(HIST1H2BK) TH1L test > 0 81 24 OR = 14.43 79.1% 82.7% 75.5% 77.1%81.3% CALM3 test ? 0 17 74 p < 2 × 10⁻¹⁶ UBE2G1 equation: −19.4675 −2.5799* ΔCt_(TH1L) + 0.9379* ΔCt_(CALM3) + 6.1263* ΔCt_(UBE2G1) TH1Ltest > 0 72 35 OR = 4.94 68.9% 73.5% 64.3% 67.3% 70.8% CALM3 test ? 0 2663 p = 1.7 × 10⁻⁷ HIST1H2BK equation: −17.0992 − 1.3345* ΔCt_(TH1L) −0.1432* ΔCt_(CALM3) + 2.3132* ΔCt_(HIST1H2BK) TH1L test > 0 84 24 OR =18.13 80.6% 85.7% 75.5% 77.8% 84.1% UBE2G1 test ? 0 14 74 p < 2 × 10⁻¹⁶HIST1H2BK equation: −23.9436 − 2.0622* ΔCt_(TH1L) + 5.8134*ΔCt_(UBE2G1) + 1.4056* ΔCt_(HIST1H2BK) CALM3 test > 0 82 25 OR = 14.7079.1% 83.7% 74.5% 76.6% 82.0% UBE2G1 test ? 0 16 73 p < 2 × 10⁻¹⁶HIST1H2BK equation: −26.9573 − 1.7284* ΔCt_(CALM3) + 4.4926ΔCt_(UBE2G1) + 2.016* ΔCt_(HIST1H2BK)

We subsequently analyzed the biomarker performance based on expressionlevels of combinations of 5 of the 6 genes tested above (Examples 2A,2B, and 3A), whose combination differentiates between normal and IBDpatients (RAP1A, BLCAP, UBE2G1, CALM3, GPX1, and NONO). Theclassification matrix and diagnostic accuracy estimates of thecombination for IBD v Normal, as well as exemplary gene weightings, areshown in Table 18. The equation and weightings for the 5-gene IBD vsNormal combination taught above (RAP1A, BLCAP, UBE2G1, CALM3, and GPX1,Example 2A) are identical to the first combination listed in Table 18.The performance measures in Tables 5a and 5b for set RAP1A, BLCAP,UBE2G1, CALM3, and GPX1) are slightly different because additionalpatients were included in the Table 18 data: Table 5a summarizes resultsfrom 280 patients, and Table 18 summarizes results from 287 patients.

TABLE 18 2 × 2 table combination IBD Normal fisher's accuracysensitivity specificity PPV NPV RAP1A test > 0 162 25 OR = 17.27 81.9%85.7% 74.5% 86.6% 73.0% BLCAP test ? 0  27 73 p < 2 × 10⁻¹⁶ UBE2G1 GPX1CALM3 equation: −10.7716 + 3.2485* ΔCt_(RAP1A) + 5.4360* ΔCt_(BLCAP) −3.4723*ΔCt_(UBE2G1) − 3.4642* ΔCt_(GPX1) + 3.3511* ΔCt_(CALM3) RAP1Atest > 0 163 32 OR = 12.78 79.8% 86.2% 67.3% 83.6% 71.7% UBE2G1 test ? 0 26 66 p < 2 × 10⁻¹⁶ GPX1 CALM3 NONO equation: −24.3638 + 3.2362*ΔCt_(RAP1A) + 0.4716*ΔCt_(UBE2G1) − 2.2953* ΔCt_(GPX1) + 2.3207*ΔCt_(CALM3) + 2.3578* ΔCt_(NONO) RAP1A test > 0 163 30 OR = 14.03 80.5%86.2% 69.4% 84.5% 72.3% BLCAP test ? 0  26 68 p < 2 × 10⁻¹⁶ GPX1 CALM3NONO equation: −22.0546 + 2.9300* ΔCt_(RAP1A) + 1.8471 *ΔCt_(BLCAP) −2.5939* ΔCt_(GPX1) + 2.0732* ΔCt_(CALM3) + 1.5925* ΔCt_(NONO) RAP1Atest > 0 160 36 OR = 9.40 77.4% 84.7% 63.3% 81.6% 68.1% BLCAP test ? 0 29 62 p = 4.3 × 10⁻¹⁶ UBE2G1 CALM3 NONO equation: −26.4580 + 4.3570*ΔCt_(RAP1A) + 2.2414*ΔCt_(BLCAP) − 1.0652* ΔCt_(UBE2G1) + 0.2504*ΔCt_(CALM3) + 1.2175* ΔCt_(NONO) RAP1A test > 0 159 25 OR = 15.28 80.8%84.1% 74.5% 86.4% 70.9% BLCAP test ? 0  30 73 p < 2 × 10⁻¹⁶ UBE2G1 GPX1NONO equation: −21.1203 + 4.7113* ΔCt_(RAP1A) + 3.8401*ΔCt_(BLCAP) −1.7575* ΔCt_(UBE2G1) − 2.2260* ΔCt_(GPX1) + 1.2861* ΔCt_(NONO) BLCAPtest > 0 165 31 OR = 14.67 80.8% 87.3% 68.4% 84.2% 73.6% UBE2G1 test ? 0 24 67 p < 2 × 10⁻¹⁶ GPX1 CALM3 NONO equation: −9.0048 +3.6181*ΔCt_(BLCAP) − 1.6280* ΔCt_(UBE2G1) − 3.6336* ΔCt_(GPX1) + 4.4814*ΔCt_(CALM3) + 0.8072* ΔCt_(NONO)

We subsequently analyzed the biomarker performance based on expressionlevels of combinations of 6 of the 7 genes tested above (Examples 2E and3C), whose combination differentiates between IBS and IBD patients(RAP1A, UBE2G1, CALM3, GPX1, HIST1H2BK1, PPP2R5A, and BLCAP). Theclassification matrix and diagnostic accuracy estimates of thecombination for IBS v IBD, as well as exemplary gene weightings, areshown in Table 19.

TABLE 19 2 × 2 table combination IBD IBS fisher's accuracy sensitivityspecificity PPV NPV RAP1A test > 0 168 27 OR = 16.26 81.5% 86.2% 72.4%85.8% 73.2% UBE2G1 test ? 0  21 71 p < 2 × 10⁻¹⁶ CALM3 GPX1 HIST1H2BKPPP2R5A equation: 16.6895 + 5.155* ΔCt_(RAP1A) − 2.5462* ΔCt_(UBE2G1) +4.8674*ΔCt_(CALM3) − 2.3979* ΔCt_(GPX1) − 2.8183* ΔCt_(HIST1H2BK) −2.9778* ΔCt_(PPP2R5A) RAP1A test > 0 167 29 OR = 17.80 82.2% 88.4% 70.4%85.2% 75.8% CALM3 test ? 0  22 69 p < 2 × 10⁻¹⁶ GPX1 HIST1H2BK PPP2R5ABLCAP equation: 13.2115 + 4.4136* ΔCt_(RAP1A) + 4.8119* ΔCt_(CALM3) −2.1923* ΔCt_(GPX1) − 3.0788* ΔCt_(HIST1H2BK) − 3.2874* ΔCt_(PPP2R5A) −1.3537* ΔCt_(BLCAP) RAP1A test > 0 168 29 OR = 18.75 82.6% 88.9% 70.4%85.3% 76.7% UBE2G1 test ? 0  21 69 p < 2 × 10⁻¹⁶ GPX1 HIST1H2BK PPP2R5ABLCAP equation: 14.9114 + 6.8342* ΔCt_(RAP1A) − 6.6508* ΔCt_(UBE2G1) −1.5818* ΔCt_(GPX1) − 1.3437* ΔCt_(HIST1H2BK) − 2.0964* ΔCt_(PPP2R5A) +4.3897* ΔCt_(BLCAP) RAP1A test > 0 171 31 OR = 20.22 82.9% 90.5% 68.4%84.7% 78.8% UBE2G1 test ? 0  18 67 p < 2 × 10⁻¹⁶ CALM3 HIST1H2BK PPP2R5ABLCAP equation: 6.224 + 7.0296* ΔCt_(RAP1A) − 5.2346* ΔCt_(UBE2G1) +1.0171* ΔCt_(CALM3) − 1.8810* ΔCt_(HIST1H2BK) − 2.1413* ΔCt_(PPP2R5A) +2.7922* ΔCt_(BLCAP) RAP1A test > 0 166 29 OR = 16.93 81.9% 87.8% 70.4%85.1% 75.0% UBE2G1 test ? 0  23 69 p < 2 × 10⁻¹⁶ CALM3 GPX1 PPP2R5ABLCAP equation: 14.1274 + 5.8613* ΔCt_(RAP1A) − 7.1962* ΔCt_(UBE2G1) +1.1262* ΔCt_(CALM3) − 2.1478* ΔCt_(GPX1) − 2.2727* ΔCt_(PPP2R5A) +4.7122* ΔCt_(BLCAP) UBE2G1 test > 0 165 39 OR = 10.29 78.0% 87.3% 60.2%80.9% 71.1% CALM3 test ? 0  24 59 p < 2 × 10⁻¹⁶ GPX1 HIST1H2BK PPP2R5ABLCAP equation: 21.6433 − 3.1122* ΔCt_(UBE2G1) + 5.9519* ΔCt_(CALM3) −3.2758* ΔCt_(GPX1) − 2.2307* ΔCt_(HIST1H2BK) − 1.2969* ΔCt_(PPP2R5A) +1.5055* ΔCt_(BLCAP) RAP1A test > 0 164 29 OR = 15.40 81.2% 86.8% 70.4%85.0% 73.4% UBE2G1 test ? 0  25 69 p < 2 × 10⁻¹⁶ CALM3 GPX1 HIST1H2BKBLCAP equation: 12.5376 + 4.5779* ΔCt_(RAP1A) − 6.7553* ΔCt_(UBE2G1) +2.5618*ΔCt_(CALM3) − 2.1679* ΔCt_(GPX1) − 1.9711 * ΔCt

1. A biomarker consisting of between 5 and 35 different nucleic acidprobe sets, wherein: (a) a first probe set consisting of one or morenucleotide probes consisting of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; (b) a second probe set consisting of one or morenucleotide probes consisting of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; (c) a third probe set consisting of one or morenucleotide probes consisting of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; (d) a fourth probe set consisting of one or morenucleotide probes consisting of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; and (e) a fifth probe set consisting of one or morenucleotide probes consisting of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; wherein each of the between 5 and 35 different probesets consists of one or more probes of 15 or more contiguousnucleotides, or full complements thereof, of a single mRNA differentfrom that of the other probe sets, and wherein each of the differentprobe sets is optionally detectably labeled.
 2. The biomarker of claim1, wherein the first probe set consists of one or more nucleotide probesof 15 or more contiguous nucleotides of RAP1A, or a full complementthereof the second probe set consists of one or more nucleotide probesof 15 or more contiguous nucleotides of BLCAP, or a full complementthereof the third probe set consists of one or more nucleotide probes of15 or more contiguous nucleotides of UBE2G1, or a full complementthereof the fourth probe set consists of one or more nucleotide probesof 15 or more contiguous nucleotides of CALM3, or a full complementthereof the fifth probe set consists of one or more nucleotide probes of15 or more contiguous nucleotides of GPX1, or a full complement thereofand a sixth probe set consists of one or more nucleotide probes of 15 ormore contiguous nucleotides of NONO or a full complement thereof.
 3. Thebiomarker of claim 2, wherein the biomarker consists of between 6 and 25probe sets.
 4. The biomarker of claim 2, wherein the biomarker consistsof between 6 and 15 probe sets.
 5. The biomarker of claim 2, wherein thebiomarker consists of between 6 and 8 probe sets.
 6. A biomarkerconsisting of between 5 and 35 different nucleic acid primer pairs,wherein: (a) a first primer pair capable of selectively amplifying adetectable portion of a nucleic acid selected from the group consistingof SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5(UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ IDNO:13 (NONO), wherein each primer in the first primer pair consists of15 or more contiguous nucleotides of a nucleic acid selected from thegroup consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP),SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1),and SEQ ID NO:13 (NONO), or a full complement thereof (b) a secondprimer pair capable of selectively amplifying a detectable portion of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO)), wherein eachprimer in the second primer pair consists of 15 or more contiguousnucleotides of a nucleic acid selected from the group consisting of SEQID NO:4 (TH1L), SEQ ID NO:9 (HIST1H2BK), SEQ ID NO:5 (UBE2G1), SEQ IDNO:3 (BLCAP), and SEQ ID NO:6 (CALM3), or a full complement thereof; (c)a third primer pair capable of selectively amplifying a detectableportion of a nucleic acid selected from the group consisting of SEQ IDNO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ IDNO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO),wherein each primer in the third primer pair consists of 15 or morecontiguous nucleotides of a nucleic acid selected from the groupconsisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP), SEQ IDNO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1), and SEQID NO:13 (NONO), or a full complement thereof; (d) a fourth primer paircapable of selectively amplifying a detectable portion of a nucleic acidselected from the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7and/or 8 (GPX1), and SEQ ID NO:13 (NONO), wherein each primer in thefourth primer pair consists of 15 or more contiguous nucleotides of anucleic acid selected from the group consisting of SEQ ID NO:1 and/or 2(RAP1A), SEQ ID NO:3 (BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3),SEQ ID NO:7 and/or 8 (GPX1), and SEQ ID NO:13 (NONO), or a fullcomplement thereof; and (e) a fifth primer pair capable of selectivelyamplifying a detectable portion of a nucleic acid selected from thegroup consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3 (BLCAP),SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8 (GPX1),and SEQ ID NO:13 (NONO), wherein each primer in the fifth primer pairconsists of 15 or more contiguous nucleotides of a nucleic acid selectedfrom the group consisting of SEQ ID NO:1 and/or 2 (RAP1A), SEQ ID NO:3(BLCAP), SEQ ID NO:5 (UBE2G1), SEQ ID NO:6 (CALM3), SEQ ID NO:7 and/or 8(GPX1), and SEQ ID NO:13 (NONO), or a full complement thereof; whereineach of the between 5 and 35 different primer pairs consists of one ormore primer pairs of 15 or more contiguous nucleotides, or fullcomplements thereof, for a single mRNA different from that of the otherprimer pairs, and wherein each of the different primer pairs isoptionally detectably labeled.
 7. The biomarker of claim 6, wherein eachprimer in the first primer pair consists of 15 or more contiguousnucleotides of RAP1A, or a full complement thereof; each primer in thesecond primer pair consists of 15 or more contiguous nucleotides ofBLCAP, or a full complement thereof; each primer in the third primerpair consists of 15 or more contiguous nucleotides of UBE2G1, or a fullcomplement thereof; each primer in the fourth primer pair consists of 15or more contiguous nucleotides of CALM3, or a full complement thereof;each primer in the fifth primer pair consists of 15 or more contiguousnucleotides of GPX1, or a full complement thereof; and each primer in asixth primer pair consists of 15 or more contiguous nucleotides of NONO,or a full complement thereof.
 8. The biomarker of claim 7, wherein thebiomarker consists of between 6 and 25 primer pairs.
 9. The biomarker ofclaim 7, wherein the biomarker consists of between 6 and 15 primerpairs.
 10. The biomarker of claim 7, wherein the biomarker consists ofbetween 6 and 8 primer pairs.